Nori Kurata

Identification of quantitative trait loci controlling heading date in rice using a high-density linkage map

Abstract Quantitative trait locus (QTL) analysis has been carried out to identify genes conferring heading date in rice. One hundred and eighty six F2 plants derived from a cross between a japonica variety, Nipponbare, and an indica variety, Kasalath, were used as a segregating population for QTL mapping and more than 850 markers were employed to identify QTLs. Scan-analysis revealed the existence of two QTLs with large effects, Hd-1 and Hd-2, one in the middle of chromosome 6 and one at the end of chromosome 7, respectively. For both loci, the Kasalath alleles reduced days-to-heading. In addition, three QTLs with minor effects, Hd-3, Hd-4 and Hd-5, were found to be located on chromosomes 6, 7 and 8 based on a secondary scan analysis which was carried out by removing the phenotypic effects of Hd-1 and Hd-2. For the three secondary loci, the Nipponbare alleles reduced days-to-heading. The five QTLs explained 84% of the total phenotypic variation in the F2 population based on a multiple-QTL model. The presence of a digenic interaction between Hd-1 and Hd-2 was clearly suggested.

Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development.

We report the isolation, sequence, and pattern of gene expression of members of the KNOTTED1 (KN1)-type class 1 homeobox gene family from rice. Phylogenetic analysis and mapping of the rice genome revealed that all of the rice homeobox genes that we have isolated have one or two direct homologs in maize. Of the homeobox genes that we tested, all exhibited expression in a restricted region of the embryo that defines the position at which the shoot apical meristem (SAM) would eventually develop, prior to visible organ formation. Several distinct spatial and temporal expression patterns were observed for the different genes in this region. After shoot formation, the expression patterns of these homeobox genes were variable in the region of the SAM. These results suggest that the rice KN1-type class 1 homeobox genes function cooperatively to establish the SAM before shoot formation and that after shoot formation, their functions differ.

Detection of segregation distortions in an indica-japonica rice cross using a high-resolution molecular map

We have constructed a high-resolution rice genetic map containing 1383 DNA markers covering 1575 cM on the 12 linkage groups of rice using 186 F2 progeny from a cross between a japonica variety, ‘Nipponbare’, and an indica variety, ‘Kasalath’. Using this high-resolution molecular linkage map, we detected segregation distortion in a single wide cross of rice. The frequencies of genotypes for 1181 markers with more than 176 genotype data were plotted along this map to detect segregation distortion. Several types of distorted segregation were observed on 6 of the chromosomes. We could detect 11 major segregation distortions at ten positions on chromosomes 1, 3, 6, 8, 9, and 10. The strongest segregation distortion was at 107.2 cM on chromosome 3 and may be the gametophyte gene 2 (ga-2). The ‘Kasalath’ genotype at this position was transmitted to the progeny with about a 95% probability through the pollen gamete. At least 8 out of the 11 segregation distortions detected here are new. The use of the high-resolution molecular linkage map for improving our understanding of the genetic nature and cause of these segregation distortions is discussed.

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.

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.

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F2 population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08750 was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).

Saturation mapping with subclones of YACs: DNA marker production targeting the rice blast disease resistance gene, Pi-b

Abstract Saturation mapping of a very small genomic region is indispensable for map-based cloning. We applied a method based on sub-cloning and the Southern-hybridization technique for generating RFLP markers directly from yeast artificial chromosomes (YACs). Two YACs overlapping each other and covering the locus of the rice blast resistance gene, Pi-b, were used to construct a plasmid sub-library. Rice-specific and single-copy clones suitable as probes for RFLP analysis were selected from this sub-library by hybridization to the blots of digested DNAs of rice, YACs, and yeast. As a result, 22 markers were produced within a small chromosomal region including Pi-b. This case study shows that overlapping YACs known to cover the gene of interest are very useful in fine-scale physical mapping leading to map-based cloning of the target gene.

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.

Expression of novel homeobox genes in early embryogenesis in rice

We isolated four novel cDNA clones of rice (Oryza sativa L.), which encode predicted proteins with a KN1-like homeodomain. In situ hybridization and RT-PCR analysis with solid cDNA libraries as templates showed that these genes are expressed in distinct patterns during the early stages of rice embryogenesis.

Organ-specific alternative transcripts of KNOX family class 2 homeobox genes of rice.

We identified three genes (HOS58, HOS59 and HOS66) of rice (Oryza sativa), which encode predicted proteins with the KNOX family class 2 homeodomain. These proteins contain three conserved domains, the KNOX domain, ELK domain and homeodomain, from an N-terminus to a C-terminus. In addition to similarity of predicted amino acid sequences, these genes showed a similar exon/intron structure. cDNA cloning and reverse transcription-polymerase chain reaction analyses of these genes indicated tissue-specific expression of alternative transcripts. The expression of the longer mRNAs of HOS58 (HOS58L) and HOS59L was detected in all organs examined such as roots, leaf blades, leaf sheaths, flowers and calli, whereas the shorter mRNAs of HOS58 (HOS58S) and HOS59S were expressed in leaf blades, leaf sheaths and flowers. The expression of HOS66L was detected in roots, leaf blades, leaf sheaths and flowers, whereas the expression of HOS66S was detected in roots and flowers. The alternative transcripts of HOS66 arose by use of alternative transcription start sites. The longer transcripts contained an exon 1 which encodes an alanine/glycine-rich region, whereas the shorter ones lacked it. These results suggest that the expression of the alternative transcripts is organ-specific, and their products have different degrees of abilities for activation or repression of transcription.