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Third International Rice Genetics Symposiumbook out of print--pdf files of
individual chapters are available after each abstract
Edited by G.S. Khush Economic prosperity in Asia: implications for rice research M. Hossain This paper describes the changing economic environment in Asia and the impacts these changes have on input-output markets and the organization of rice production in high- and low-income countries. Emerging problems in Asian rice farming related to sustaining farmers' incentives in high-income countries and food security in low-income countries are discussed. The author assesses the priorities for genetic rice research that will be required to develop appropriate technologies needed to address the problems. These priorities are grain quality, resistance to abiotic and biotic stresses, shifting the yield frontier, direct seeding for crop establishment, and incorporating micronutrients. R.W. Herdt This paper presents the methodology and results of a procedure used to establish the funding priorities of the Rockefeller Foundation support for rice biotechnology research. Data from a wide range of sources, including new national-level research on rice production losses, are used, together with data on area of land planted to each of the four main rice agroecologies in Asia, Latin America, and Africa, current yields, and estimates of the relative difficulty of achieving various research goals. Priorities are derived by simultaneously considering both the relative importance of constraints and the relative difficulty of overcoming the constraints. Approximately US$443 million could be invested annually in rice biotechnology re search to achieve an internal rate of return of 35%, allocated across five categories of challenges and four agroecologies in the developing world. High-density linkage map of rice with expressed sequence tags B.A. Antonio, A. Shomura, T. Shimano, Y. Kuboki, S.Y. Lin, T. Inoue, H. Kajiya, Y. Harushima, K. Yamamoto, Y. Nagamura, N. Kurata, M. Yano, Y. Minobe, and T. Sasaki We have constructed a high-density linkage map of rice using an F 2 population derived from the cross between a japonica variety, Nipponbare, and an indica variety, Kasalath. A total of 1,383 markers, which consist of cDNA clones from callus and root, genomic clones as well as RAPD markers, have been mapped covering a distance of 1,575 cM. All cDNA clones have been sequenced and searched for similarities with known proteins and can be referred to as expressed sequence tags on the map. A majority of the genomic clones and RAPD markers was also sequenced to generate sequence-tagged sites. These extensive linkage analyses gave evidence on duplication of chromosomal segments, particularly in the distal region of chromosomes 11 and 12. Additional markers are being mapped using cDNA clones derived from other cDNA libraries such as green shoot, etiolated shoot, and developing seed. Ultimately, we would like to develop a saturated linkage map that will facilitate a more efficient utilization of molecular markers for rice improvement. An integrated linkage map of rice O. Ideta, A. Yoshimura, and N. Iwata To furnish significant information for genome analysis of rice, we have tried to integrate the morphological and restriction fragment length polymorphism (RFLP) linkage maps of all chromosomes. Thirty -nine morphological markers and 82 RFLP markers were mapped together based on segregation analysis of 19 F2 populations derived from the crosses between indica variety IR24 and japonica lines with different morphological markers. Both linkage maps of rice were completely orientated. The gene d2, which had been thought to be long to the linkage group of chromosome 4, was linked to some RFLP markers on chromosome 1. Therefore, chromosomal location of d2 was shifted to chromosome 1 from chromosome 4. Unmapped genes gl1 on chromosome 5 and I-Bf on chromosome 9 were mapped on their respective linkage groups with some RFLP markers. The segregation distortion was detected on chromosomes 1, 2, 3, 6, 11, and 12. In these regions, the japonica/japonica allele frequencies were significantly less than the normal F2 frequency of 25%. The RFLP linkage maps on chromosomes 2, 3, 6, 7, and 8 were considered not to be saturated. On these chromosomes, some marker genes were estimated to be located beyond the respective terminal ends of RFLP linkage maps. Centromere mapping and orientation of the cytological, classical, and molecular linkage maps of rice G.S. Khush, K. Singh, T. Ishii, A. Parco, N. Huang, D.S. Brar, and D.S.Multani IRRI developed a series of secondary trisomics and telotrisomics and used these for assigning genes and restriction fragment length polymorphism markers to specific chromosome arms and mapped position of centromeres on both classical and molecular linkage maps. Corrected orientations of both the classical and molecular linkage maps were determined and a linear correspondence among the cytological, classical, and molecular maps was established. Rice as the pivotal genome in the new era of grass comparative genetics M.D. Gale, K.M. Devos, and G. Moore Comparisons of the molecular maps of a number of grass species, including the major cereals rice, wheat, and maize, indicate that gene linkage relationships have been remarkably maintained ever since isolation by speciationas long as 60 million yr ago. Collinearity between the maps of six grass crop species is such that a single consensus genetic map can be described by only 19 rice linkage blocks. These findings will be translated into the use of rice as a tool for cross genome map-based gene isolation, transfer and synthesis of information concerning syntenous genes and biochemical, developmental, or physiological pathways over species, and the availability of a wide range of alternative homoeogenes for the transgenic cereal breeder of the next century. Construction of a first-generation physical map of the rice genome I. Ashikawa, Y. Umehara, H. Tanoue, Z. Wang, S. Saji, T. Shimokawa, K. Yoshino, J. Wu, B. Antonio, K. Koike, W. Van Houten, A. Idonuma, M. Emoto, N. Kurata, and T. Sasaki We aim to construct a physical map of rice consisting of overlapping yeast artificial chromosome (YAC) clones that span all of the rice chromosomes. Using 1,383 restriction fragment length polymorphism markers and sequence-tagged sites established on our rice linkage map, we have started to screen our rice YAC library, which contains 6,932 rice YAC clones and represents six genome equivalents. As of August 1995, we have used 1,126 markers for the screening. Of these, 1,004 markers hybridized with a total of 4,645 YAC clones in the library. Out of these YACs, 2,369 were independent. We matched the locations of the isolated YACs to corresponding positions on the rice linkage map, detected overlaps between the YACs, and assembled all YACs to contigs. About half of chromosome 6 has now been covered with YAC contigs. We expect that such a combined genetic/physical map will provide a basis for analyzing the genome structure and for map-based cloning of agronomically important genes. Application of cDNA sequence information for characterization of the rice genome A. Miyao, K. Yamamoto, C. Hamamatsu, H. Itadani, Y. Koga-Ban, K. Koike, S. Kojima, H. Nagasaki, T. Shimizu, Y. Shinozuka, J. Song, U. Yamanouchi, M. Yoshikawa, L. Monna, H. Sun Zhong, H. Ichimura, and T. Sasaki We have sequenced more than 20,000 clones from rice cDNA libraries up to Apr 1995. The libraries were made from shoot, root, and callus treated with various hormones or with a heat shock. Functions of the predicted gene products were deduced by similarity search against the NBRF-PIR data base using the FASTA algorithm. Profiles of gene expression (judged by frequencies of cDNAs coding for various genes) in these libraries closely reflected the physiological state of the rice tissue examined. The majority of proteins identified from green shoot cDNAs were photosynthesis-related, totaling more than 10% of the total number of clones. In etiolated shoots, it was rare to find these cDNA clones. In the normal callus library, the most often observed homology hits were translation-related proteins, histone proteins, etc. In the heat shock callus library, the most common hit clones had nucleotide sequences similar to heat shock proteins. From our genomic nucleotide sequence data of 1,220 clones and cDNA sequence data, we calculated frequencies of all decanucleotide sequences. In rice cDNA, after poly-A, the most abundant repetitive sequence is the CCG repeat. Repeats of AGG, AGC, AAG, ACC, ACG, and AG also appeared with high frequencies. We chose decamer nucleotide sequences for reliable RAPD primers using our decamer frequency data. Primers that have low frequency sequences could amplify several DNA fragments to detect clear polymorphisms. Structure and gene expression of the mitochondrial genome of rice M. Nakazono, N. Tsutsumi, and A. Hirai We examined the structure and organization of the mitochondrial genome and the expression of the mitochondrial genes of rice ( Oryza sativa cv Nipponbare). In particular, the entire set of the transferred chloroplast DNA sequences and a family of the palindromic repeated sequences that mainly exist in the intergenic spacer regions were identified. Furthermore, we determined the transcription initiation sites of rice mitochondrial genes by in vitro capping and ribonuclease protection assays and primer extension analysis. A consensus sequence was observed around the sites of initiation of transcription, indicating that the consensus motif is recognized as promoters of rice mitochondrial genes. Advances in rice chromosome research, 1990-95 K. Fukui After providing a historical perspective on rice chromosome research, this paper reviews and summarizes advances made during the 1990 -95 period. The most significant research accomplishments during the last 5 yr are the complete and objective identification of all the somatic rice chromosomes (80 yr after the first accurate report of the number of rice chromosomes) and the development of a quantitative rice chromosome map. Also, in 1991, the long discussion on unifying the various numbering systems for the rice chromosomes ended with the implementation of a new numbering system. New technologies recently developed include 1) imaging methods that make it possible to identify rice chromosomes automatically using personal computers, 2) fluorescence in situ hybridization, 3) genomic in situ hybridization for differential painting of the rice genomes, and 4) the laser microdissection method used in dissecting specific regions of individual rice chromosomes. Stable inheritance of transgenes in rice plants transformed by Agrobacterium tumefaciens Y. Hiei and T. Komari Inheritance of foreign genes in rice transformed by Agrobacterium tumefaciens has been investigated up to the R 4 progeny. Rice cultivar Tsukinohikari was transformed with A. tumefaciens strains LBA4404(pTOK233) and EHA101(pIG121Hm). Cultivar Koshihikari was transformed with LBA4404(pTOK233). pTOK233 is a "super -binary" vector and LBA4404(pTOK233) had a greater transformation efficiency than EHA101(pIG121Hm). The T-DNA of these strains carried a gus gene and a hygromycin resistance gene, both of which were driven by the 35S promoter. The gus gene contained an intron in the coding region. The selfed progeny of 20 independent plants from each of the three populations of transformants were evaluated for gus expression and resistance to hygromycin. The two markers were genetically linked and inherited in a Mendelian fashion. The segregation ratio of 3:1 (positive:negative for the transgenes) was observed in the progeny of 60-75% of the transformants in each of the groups of 20 plants. The R2 progeny of six plants from each of the groups were scored for GUS expression and hygromycin resistance. Thus, the Mendelian transmission of the two markers was further confirmed and offspring homozygous for the two genes were identified in the R2 lines. R3 plants were obtained from the homozygous R2 offspring, and consequently R4 plants were produced. The two genes were expressed in the R3 and R4 generations and none of the plants in these generations lost the genes. The DNA from selected offspring was analyzed by Southern hybridization and a tight correlation between the phenotype and genotype was demonstrated. Agrobacterium-mediated transformation of an elite cultivar of Texas rice J.J. Dong, W.M. Teng, W.G. Buchholz, and T.C. Hall While useful advances have been made using biolistic and other naked DNA uptake methods for rice transformation, high copy numbers of rearranged sequences are common and lack of stable expression due to silencing or other causes is proving to be a major problem. Attempts to transform rice and other monocots using Agrobacterium tumefaciens-based vectors have typically yielded results that were promising, but inconclusive. Recently, an Agrobacterium-based system starting from scutellar tissue has been described in the literature. Using only minor modifications of this system, we were able to repeat their transformation of callus de rived from the cultivar Koshihikari. We have also extended the work to include Gulfmont, a javonica variety widely used for commercial cultivation in Texas, and Taipei 309, a japonica line that has been widely used for transformation by direct DNA transfer. Transformation using the Agrobacterium approach occurred at a reasonably high frequency and vigorous, phenotypically normal plants were recovered. Only one or two unrearranged copies of the T-DNA were present at a single locus; no silencing of the selectable marker or the gene of interest (gus) was observed and the introduced genes were transmitted in a normal Mendelian fashion. Biolistic transformation of rice: now efficient and routine for japonica and indica rices C.M. Fauquet, S. Zhang, L. Chen, P. Marmey, A. de Kochko, and R.N. Beachy Rice transformation using microbombardment has been used for 4 yr, but it is only since 1994 that we can consider the process routine for both japonica and indica rices. For japonica transformation, the rate of efficiency of transformation now averages 25%. Our protocol does not generate escapes for hygromycin resistance and the duration of our protocol has been shortened to 8 wk from shooting time to obtaining plantlets. Transformation is accomplished by mixing plasmids containing the hygromycin resistance gene and the gene(s) of interest (GOI). Cointegration of two genes averages 70% and greatly depends on the DNA ratio in the plasmid mixture. Several types of explant tissues such as immature embryos, embryogenic calli, and embryogenic suspensions have been tested and transgenic plants were produced. Embryogenic calli are preferred but, in some cases, embryogenic suspensions can be advantageous. Indica transformation using embryogenic suspensions and the biolistic method is performed regularly with varieties that are difficult to regenerate such as IR72, IR64, and BG90-2 but with a lower efficiency ranging from 1 to 5%. Transgenic plants have been carried through to seven generations, which proves stability in the inheritance of the integrated genes and shows that normal segregation is maintained over generations. Many different GOIs have now been inserted, including the Xa21 bacterial blight resistance gene, demonstrating that large pieces of DNA coding for large proteins can be successfully integrated and expressed in rice and can produce the expected phenotype. R. Wu, X. Chen, B. Wang, M. Chang, D. Xu, X. Duan, and P. Chen Transformation of rice cells followed by regeneration of fertile plants constitutes a new method for producing agronomically useful transgenic rice plants. We have transformed rice with a plasmid harboring a potato protease inhibitor gene (PINII ) joined to a wound-inducible pin2 promoter. The transgenes in plants of the R 0, R1, and R2 generations have been analyzed, and the results showed that the transgene is stably inherited. Transgenic plants have also proven to be useful in analyzing the expression of a gene or a modified gene. Transfer of genes into rice cells, followed by determining the amount of the gene products several hours later, constitutes a convenient way to analyze transient gene expression. We have used this transient assay to look for gibberellin (GA)-responsive sequences in GA-induced genes such as the rice a-amylase gene, Osamy-c. In this assay, different lengths of the promoter region were fused to a reporter gene such as the luciferase cDNA. Several plasmids were constructed and transferred into rice aleurone cells by the biolistic method, and the luciferase activity was measured. We have also used the transient assay system to look for intermediates in the GA-dependent signal-transduction pathway by transactivation experiments. These involve transferring two genes on two separate plasmids together into rice cells and observing how the gene product of the first gene affects the expression of the second. By using this approach, we have shown that a ubiquitin-conjugating enzyme gene and a calcium-dependent ATPase gene are putative intermediates in the GA-dependent signal-transduction pathway because either gene can transactivate the expression of a target gene, Osamy-c. Identification of other putative intermediates in the GA-signaling pathway is also presented. I. Potrykus, G.A. Armstrong, P. Beyer, S. Bieri, P.K. Burkhardt, H. Ding Chen, G.C. Ghosh Biswas, S.K. Datta, J. Fütterer, A. Klöti, G. Spangenberg, R. Terada, J. Wünn, and H. Zhao Indica rice provides the staple food for more than 2 billion poor in developing countries. In humid and semihumid Asia where rice is the basic food, the population is expected to increase by 58% over the next 35 yr. Increased yields to meet the demand for more food may come from reducing the present 40% harvest losses attributed to biotic and abiotic stresses. We focus on developing disease and insect resistances and improved nutrition quality through the use of genetic engineering. Toward this goal, we have a) developed a gene transfer technology for indica rice, b) introduced numerous genes that code for proteins with antifungal activity with an initial success in increasing resistance to sheath blight, c) produced cryIA(b)-transgenic IR58 with good resistance to yellow stem borer and other lepidopteran insect pests, d) regenerated numerous transgenic offspring populations that harbor a series of RTBV-DNA sequences representing several anti-tungro disease strategies, and e) activated the terpenoid pathway with an endosperm-specific, transgenic phytoene synthase such that the endosperm synthesizes good quantities of phytoene for further (transgenic) conversion to ß -carotene. A series of additional experiments include approaches toward nitrogen fixation, enhanced phosphate uptake, improved iron availability, and apomictic stabilization of hybrid vigor. Genetic engineering of rice for resistance to homopteran insect pests J.A. Gatehouse, K. Powell, and H. Edmonds The rice brown planthopper (BPH; Nilaparvata lugens ) is a serious pest of rice crops throughout Asia, damaging plants both through its feeding behavior and by acting as a virus vector. Like many homopteran pests of crops, it is primarily a phloem feeder, abstracting sap via specially adapted mouthparts. An artificial diet bioassay system for this pest was developed to allow the effects of potentially insecticidal proteins to be assayed. Several lectins and oxidative enzymes were found to be toxic to BPH. Snowdrop ( Galanthus nivalis) lectin (GNA) was selected for further study as it is nontoxic to higher animals. A cDNA encoding GNA was assembled into constructs for expression in transgenic plants, with the aim of producing transgenic rice plants that would express the foreign protein in their phloem sap and be resistant to BPH. Constitutive expression of GNA in model plant systems was shown to have deleterious effects on the development of lepidopteran and homopteran pest insects. Phloem-specific promoters for expressing GNA in transgenic rice were isolated and characterized with the aim of increasing the effectiveness and specificity of the protection against BPH. A construct containing the GNA coding sequence driven by the promoter from the rice sucrose synthase RSs1 gene was tested in tobacco and transformed into rice. Transgenic rice plants containing this construct are currently being evaluated. Construction of transgenic rice plants resistant to rice yellow stunt virus, a plant rhabdovirus R.-X. Fang, H.-T Zhu, Q. Wang, K.-Q. Mang, D.-M. Gao, W.-S. Qin, L. Zhang, S.-Y. Cao, W.-Z. Tian, and L.-C. Li Rice yellow stunt is an important disease found in China and some other Asian countries. The pathogen of the disease was identified as a rhabdovirus, which contains a single-stranded, negative-sense RNA molecule as its genome. We have cloned and completely deter mined the nucleotide sequence of the gene coding for the viral nucleocapsid protein, a structural protein associated with the viral genome. To develop a novel strategy against the infection of plant rhabdoviruses, a cDNA copy of the rice yellow stunt virus (RYSV) nucleocapsid protein gene, or a reading frame-shift mutant of the gene has been inserted into a rice expression vector under the control of the rice Act1 promoter. The constructs have been introduced into rice genomes of two RYSV-susceptible cultivars, Xiushui 11 and Bing 88, both japonicas, by an improved biolistic method. Numerous rice plants have been regenerated. Virus resistance assays were performed on 23 plants from seven independent primary transgenic plant lines by using viruliferous rice leafhoppers. The preliminary results showed that plants transformed with both constructs displayed resistance to the RYSV infection, indicating that this resistance might be mediated by the RNA transcripts of the nucleocapsid protein gene. FLP/FRT-mediated manipulation of transgenes in the plant genome T.K. Hodges, L.A. Lyznik, K.V. Rao, H. Kononowicz, K.S. Rathore, and R. Su Site-specific recombinases catalyze recombination reactions between two nucleotide sequences referred to as recombination sites. If such recombination sites are integrated into genomic DNA, depending on the orientation of these sites relative to each other, i.e., direct or inverted, the intervening genomic DNA sequence can be either inverted or excised by certain site-specific recombinases. If the recombination sites are on two different chromosomes, some of the recombinases can catalyze the exchange of chromosomal fragments. Thus, site-specific recombination reactions have the potential to have substantial practical applications in genetic engineering. We have investigated one of these site-specific recombinases, the FLP/ FRT system from the 2-µm plasmid of Saccharomyces cerevisiae , with respect to its function in cells of monocots, especially maize and rice. We have shown that the FLP recombinase can recognize and recombine FRT sites located in a plasmid molecule in maize or rice protoplasts, and if the FRT sites are on two different plasmids, they can be recombined by FLP. A recombinase test target vector containing FRT sites (pUFNeoFmG), the complete sequence being ubiquitin promoter/FRT site/neo gene/a mutated FRT site/gusA gene, was introduced into protoplasts. Calli were selected on kanamycin and suspension cultures were made. Protoplasts of these suspension cultures were treated with a FLP expression vector (pUbiFLP) containing the maize ubiquitin promoter driving FLP. ß-glucuronidase (GUS) activity resulting from the recombination-mediated excision of neo (resulting in activation of gusA ) was observed in 3-4% of all treated protoplasts. Southern blot analyses of putatively retransformed calli (GUS+) indicated that all of the calli contained the product of the site-specific recombination reaction. Protoplasts from the same suspension culture were also treated with pUbiFLP and pHyg (containing the hygromycin resistance gene), and calli were selected on hygromycin. A recombined FRT site in the genome of one of the selected GUS+ callus lines was sequenced, which confirmed that the recombination reaction indeed produced the chimeric FRT/FRTm site. To eliminate the second retransformation step in the genomic DNA excision procedure, the target vector, pUFNeoFmG, was cointroduced into maize protoplasts together with the FLP gene driven by a soybean heat-shock promoter. FLP expression was activated (based on Western blots) in transgenic calli by a heat -shock treatment. Some of these heat-shocked transgenic calli had also lost NPTII activity and gained GUS activity. Southern blot analyses indicated that the expected recombination product had been produced; however, not all the neo genes had been excised from the genome. This was due to the initial integration of multiple copies of the target vector (intact or fragmented) into the genome. P. Christou, P. Vain, A. Kohli, M. Leech, J. Oard, and S. Linscombe Transgenic rice plants from indica, japonica, and javanica varieties that express several foreign genes were generated via particle gun bombardment of immature embryos. Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression, and stability of integrated DNA through multiple generations. Such evaluations were carried out in the greenhouse (five generations) and in the field (three generations). Stability of foreign DNA was found to be dependent on the nature of the promoter and the transgene, and in specific cases, on gene copy number. A general conclusion of the results presented here is that direct DNA transfer utilizing electric discharge particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety -independent fashion. Production and testing of insect-resistant transgenic rice plants Q. Xue, X. Duan, D. Xu, and R. Wu Suspension cells or calli from three varieties of rice ( Oryza sativa L.) were transformed, by microprojectile bombardment, with plasmids that contain the bacterial phosphinothricin acetyl transferase (PAT) gene (bar), and the potato protease inhibitor II (PINII) gene or the cowpea trypsin inhibitor (CpTi) gene. Fourteen transgenic lines were grown in an isolated experimental field at Zhejiang Agricultural University in Hangzhou. Rice plants were tested for their resistance to herbicide (1% BastaTM) and two species of stem borer ( Chilo suppressalis and Sesamia inferens). The leaves of most transgenic rice plants were not affected by the herbicide, but the leaves of all nontransformed control plants became yellow. Infestation of the rice plants by the stem borers caused severe damage to nontransformed control plants and resulted in dead panicles (commonly known as whitehead). In contrast, transgenic rice plants showed much less damage and fewer dead panicles. Thus, introduction of an insecticidal proteinase inhibitor gene into rice plants appears to be a useful strategy for controlling insect pests. The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein P. Ronald, W.-Y. Song, G.-L. Wang, L.-L. Chen, H.-S. Kim, L.-Y. Pi, T. Holsten, B. Wang, C. Williams, W.-X. Zhai, L.-H. Zhu, and C. Fauquet A major goal of plant genome research is to develop and implement strategies for the isolation and engineering of agronomically important traits. The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by map-based cloning. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice. Molecular analysis of the interaction between Xanthomonas oryzae pv. oryzae and rice F.F. White, J.M. Chittoor, J.E. Leach, S.A. Young, and W. Zhu Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease of rice in Asia. Future genetic strategies to improve resistance will benefit from a better understanding of the molecular events that mediate resistance in the interaction of host and pathogen. We have characterized two avirulence genes, avrXa10 and avrXa7 , which trigger resistance in rice lines carrying the resistance genes Xa10 and Xa7, respectively. The two avr genes are members of an avirulence gene family that appears to be exclusive to Xanthomonas. In efforts to further under stand the modes of action of the genes, the interdependence of avr and hrp gene function is being investigated. Activity of avrXa10 requires a functional hrp regulon. The dependence does not appear to involve avr gene regulation or lack of bacterial growth in hrp mutants, and the hrp genes cannot rescue avr activity when supplied in planta by mixed bacterial strain inoculation. The results suggest that avr activity requires that both type of genes be present in the same bacterial cell. The hrp secretory pathway may be required for avr protein secretion or for secretion of an elicitor whose synthesis is catalyzed by avr protein products. At the same time, peroxidase and related host genes that are induced during a resistance reaction are being characterized. Cationic peroxidase PO-C1 was localized to xylem lumen and parenchyma cell walls at the site of infection by an incompatible strain of Xoo. Peroxidase transcript levels appear to peak within 12 h of challenge with bacteria. Exploring the application of molecular markers for improving resistance to bacterial blight R.J. Nelson, E. Ardales, M. Baraoidan, I. Yap, M.L.C. George, D.H. Chen, M. Finckh, A. Bordeos, C.M. Vera Cruz, T. Adhikari, C.C. Mundt, M. Bustamam, W. Cruz, H. Leung, N. Huang, A. Yoshimura, S. McCouch, T.W. Mew, and J.E. Leach Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is endemic in many parts of Asia and periodic epidemics can cause severe yield losses. Although many genes for resistance to bacterial blight are known, few have been systematically used for rice improvement. To allow the judicious use of resistance, it is important to understand the population biology of the pathogen and to understand the genetics of resistance. Molecular marker technology has contributed to both of these objectives. A series of molecular markers was developed for analysis of Xoo populations, including four transposable elements, a family of avirulence genes, and a set of polymerase chain reaction-based markers. One of these primers can be used for DNA fingerprinting of pathogen cells oozed directly from infected leaves. To shed light on the diversity and distribution of Xoo, a hierarchical analysis of variation was conducted. Pathogen populations from different Asian countries were distinct. Intensive sampling was conducted in the Philippines, revealing population differentiation between regions, sites, and even fields within a site. Certain DNA fingerprint types (haplotypes) were widely distributed (even between countries), providing evidence of pathogen movement. By subsampling the dataset, an optimized sampling strategy could be recommended for subsequent studies. Resistance genes for bacterial blight have been tagged and mapped in several laboratories. Using restriction fragment length polymorphism-based methodology, even genes with minor effects on disease (putative quantitative trait loci) have been detected. Based on inoculation data, gene combinations likely to be effective for disease reduction in the field were designed. Several rice lines carrying multiple genes have been produced by marker-assisted selection. In some cases, such as in the Xa4 + xa5 line, plants carrying pairs of resistance genes show greater resistance than expected based on the performance of the individual genes. Experiments on the deployment of resistance genes are being conducted in Laguna, Philippines. These studies are making use of near-isogenic lines carrying different resistance genes, as well as mixtures and pyramid lines. Analysis of pathogen subpopulations sampled from different treatments indicated which genes are functionally distinct in this environment. Lines carrying xa5, expected to be resistant, showed low levels of disease due to selection of a rare pathogen lineage. Now that powerful and inexpensive methods are available for analyzing pathogen populations and for identifying resistance loci, it will be possible to deploy resistance more effectively. Molecular analysis of repeated DNA sequences from the rice blast fungus Magnaporthe grisea P. Kachroo, S.A. Leong, and B.B. Chattoo Genome organization and distribution of repeated DNA sequences were studied in the rice blast fungus Magnaporthe grisea . The analysis led to the identification of an inverted repeat transposon and a short interspersed nuclear element. Both of these elements were isolated as insertions into unrelated repeated DNA sequences, a process that appears to be of common occurrence in the genome of M. grisea. The repeated DNA sequences were characterized with respect to their fingerprinting patterns in rice and nonrice isolates of M. grisea, dispersion in the genome, copy number, and sequence characteristics. The close physical association of diverse repeated DNA sequences and the genome rearrangements caused by them indicate that they may also contribute to genome variability seen among isolates of M. grisea. Sequence-tagged sites and low-cost DNA markers for rice J.A. Robeniol, S.V. Constantino, A.P. Resurreccion, C.P. Villareal, B. Ghareyazie, B.-R. Lu, S.K. Katiyar, C.A. Menguito, E.R. Angeles, H.-Y. Fu, S. Reddy, W. Park, S.R. McCouch, G.S. Khush, and J. Bennett Sequence-tagged sites (STSs) facilitate the conversion of a genetic map into a physical map, provide a common basis for the comparison of diverse types of mapping data, are stored and disseminated as electronic data, and are amplified from genomic DNA by polymerase chain reaction (PCR). STSs find application as DNA markers in breeding programs and germplasm management because they offer speed, convenience, reliability, and low cost in genomic analysis, but these applications are currently limited by the small number of STSs available. We report here the terminal sequencing of 354 DNA markers of the Cornell-IRRI genetic map of rice and the conversion of 100 of them into STSs by synthesis of pairs of PCR primers. PCR was used to amplify the corresponding loci from genomic DNA of IR36 (indica), Taichung 65 (japonica), and Oryza longistaminata (AA genome wild species). More than half of the RZ clones amplified DNA segments that were 0.1-2.0 kbp larger than expected, presumably because of the presence of introns. Amplicon length polymorphisms were detected between O. sativa and O. longistaminata for about one quarter of the clones. The applications of STSs are illustrated by reference to 1) DNA marker-aided selection for pyramiding of bacterial blight resistance genes, 2) breeding for gall midge resistance, 3) monitoring the inheritance of transgenes, and 4) analysis of genetic variation of AA genome wild species. Marker-assisted prediction of agronomic traits using diverse rice germplasm P.S. Virk, B.V. Ford-Lloyd, M.T. Jackson, H.S. Pooni, T.P. Clemeno, and H.J. Newbury A set of 47 diverse accessions of Asian rice was grown in a single trial at IRRI. The number of culms per plant and the time to 50% flowering were measured. DNA was then extracted and a set of 63 randomly amplified polymorphic DNA (RAPD) markers that were polymorphic across the 47 accessions was scored. Multiple regression analysis was carried out to determine whether associations between the presence/absence of individual markers and performance for the two quantitative traits could be detected. The results showed that 85% of the variation for culm number and nearly all the variation in flowering time could be explained by regression models using sets of RAPD markers. Isozyme analyses were then carried out on the same 47 accessions yielding a set of 39 polymorphic markers. Multiple regression analysis revealed that 70% of the variation in culm number and 49% of the variation in flowering time could be explained using this more limited set of markers. Analyses were performed using RAPD and isozyme data together to deter mine the best regression models and for culm number an improvement of 5% in r2 was achieved. Using these models for the remaining 46 accessions, predictions were made of performance for the two traits for each of the 47 accessions individually. Only four of the 94 performance predictions significantly differed from the observed performance. Relationships between molecular marker polymorphism and hybrid performance in rice Q. Zhang, M.A. Saghai Maroof, G.P Yang, K.D. Liu, Z.Q. Zhou, K.A. Gravois, C.G. Xu, and Y.J. Gao This study investigated the relationship of molecular marker heterozygosity with hybrid performance and heterosis in rice in an attempt to make use of molecular markers in predicting hybrid performance. Four sets of diallel crosses, 147 in total, were made using parents encompassing a wide range of germplasm, including 1) a set of 28 crosses using 8 elite parental lines commonly used in hybrid rice production in China, 2) a set of 36 crosses using 9 indica varieties representing a wide range of germplasm from three countries, 3) a set of 55 crosses using 11 japonica varieties also with a wide range of representation from three countries, and 4) a set of 28 crosses using 8 long-grained rice lines from the southern United States. All F1s and their parents were evaluated for agronomic performance in replicated field trials. The parental lines were surveyed for DNA polymorphisms using more than 100 molecular markers including restriction fragment length polymorphisms and simple sequence repeats. The marker genotypes of the F1s were deduced from the genotypes of their parents. The results showed that there are extensive genetic polymorphisms among the parental lines of each diallel set. High levels of heterosis are common in yield and yield component traits in crosses of all four diallel sets. The correlations between marker genotype divergence and hybrid performance varied greatly from one data set to another depending on the materials studied. The highest correlation was observed between hybrid yield and general heterozygosity in the set of southern U.S. long-grained varieties. A high level of correlation was also observed between specific heterozygosity and heterosis in the set of Chinese hybrid rices, whereas the correlations were much lower in the sets of indica and japonica mixtures. Detailed characterization of the rice germplasm and in-depth understanding of the genetic basis of heterosis are needed for molecular markers to have practical utility in predicting hybrid performance. Dominance as the major genetic basis of heterosis in rice J. Xiao, J. Li, L. Yuan, and S.D. Tanksley A set of 194 F7 lines derived from a subspecific rice cross showing strong F1 heterosis was backcrossed to the two parents to produce BC1F7 families. The 388 BC1F7 families and the 194 F 8 lines along with the two parents and their F1 were evaluated for 12 quantitative traits. Thirty-seven significant quantitative trait loci (QTLs), with a log of odds > 2.0, were detected in the BC1F 7 populations through 141 restriction fragment length polymorphism markers covering approximately 95% of the rice genome. Twenty-seven (73%) QTLs were detected in only one of the two backcross populations. In 82% of these cases, the heterozygotes were superior to the respective homozygotes. The remaining 10 (27%) QTLs were detected in both backcross populations. In all of these cases, the heterozygote had a phenotype falling between those of the two respective homozygotes and in no instances were the heterozygotes found to be superior to either of the homozygotes. These results suggest that dominance complementation (including partial dominance) is the major genetic basis of heterosis in rice. Genetic and molecular analysis of the anthocyanin pigmentation pathway in rice A.R. Reddy The anthocyanin pigmentation in rice is an example of many dispersed genes controlling a single trait, the purple/red color. We are interested in unraveling the genetic and molecular basis of expression and regulation of genes of the anthocyanin pathway for their eventual manipulation toward resistance of rice plants to various stresses, particularly diseases. Genetic analysis of phenotypic variation in tissue-specific accumulation of anthocyanins among different indica rice lines was performed. A dominant inhibitor gene, named Ilb, which inhibits pigmentation in the leaf blade, was uncovered. The major anthocyanidin pigment in rice was identified as cyanidin and the minor one as peonidin. The major proanthocyanidin in rice was tentatively identified as procyanidin. Molecular cloning of rice genes concerned with flavonoid and anthocyanin production has been achieved by using the corresponding cDNA sequences of maize as probes. A cDNA library, made from the leaf poly A + RNA preparation of young Purpleputtu (PP) seedlings, was screened with maize cDNA sequence probes. The gene-specific clones were isolated, restriction-mapped, and sequenced. The salient features of a rice cDNA clone named OsCHS (chalcone synthase), which hybridizes to the maize c2 cDNA, were described. Sequence comparison and Western analysis revealed an extensive homology between the rice and maize CHS cDNA and protein. The CHS locus of rice has been mapped to chromosome 11 using the restriction fragment length polymorphism approach. We have demonstrated that the anthocyanin biosynthesis in rice, particularly PP, is ultraviolet (UV) light-responsive. The abundance of the anthocyanin gene-specific transcripts in mRNA preps from UV -induced seedlings has been demonstrated. Molecular cloning and characterization of genes of the anthocyanin pathway, coupled with the availability of reliable and relatively easier genetic transformation methods in indica rice, offer unique advantages in genetic engineering of the pathway to unambiguously prove the role of flavonoids in resistance to disease and abiotic stresses as well. D.G. Muench, Y. Zhang, Y. Wu, and T.W. Okita Electron microscopic studies reveal that rice endosperm cells contain two types of endoplasmic reticulum (ER), the cisternal ER (C-ER) and protein body ER (PB-ER). As demonstrated by biochemical techniques and in vitro and in situ hybridization, mRNAs that code for the prolamine storage proteins are enriched up to 10-fold over the mRNAs that code for the glutelin storage proteins on the PB-ER, whereas glutelin mRNAs are about 2-fold more abundant than prolamine mRNAs on the C-ER membranes. Therefore, the initial targeting of glutelins and prolamines into separate protein bodies is facilitated by the segregation of their transcripts on the two distinct ER types. We have obtained evidence suggesting that at least one process that is responsible for targeting of the prolamine mRNA to the PB-ER is the interaction of the prolamine transcript with a receptor on the PB surface. In addition to these studies, we discuss our current efforts to obtain more direct evidence for the mechanism of sorting by generating transgenic plants containing hybrid gene constructs. |
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Economic prosperity in Asia: implications for rice research Establishing the Rockefeller Foundation's priorities for rice biotechnology research in 1995 and beyondHigh-density linkage map of rice with expressed sequence tags An integrated linkage map of rice Centromere mapping and orientation of the cytological, classical and molecular linkage maps of rice Rice as the pivotal genome in the new era of grass comparative genetics Construction of a first-generation physical map of the rice genome Application of cDNA sequence information for characterization of the rice genome Structure and gene expression of the mitochondrial genome of rice Advances in rice chromosome research, 1990-1995 Stable inheritance of rice gened transformed by Agrobacterium tumefaciens Agrobacterium-medical transformation of an elite cultivar of Texas rice Biolistic transformation of rice: now efficient and routine for japonica and indica rices Genetic engineering of rice plants for resistance to homopteran insect pests Construction of transgenic rice plants resistant to yellow stunt virus, a plant rhabdovirus FLP/FRT-mediated mainpulation of transgenes in the plant genome Production and testing of insect-resistant transgenic rice plants The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein Molecular analysis of the interaction between Xanthomonas oryzae pv. oryzae and rice Exploring the application of molecular markers for improving resistance to bacterial blight Molecular analysis of repeated DNA sequences from the rice blast fungus Magnaporthe grisea Sequence-tagged sites and low cost DNA-markers for rice Marker-assisted prediction of agronomic traits using diverse rice germplasm Relationships between molecular marker polymorphism and hybrid performance in rice Dominance as the major genetic basis of heterosis in rice Genetic and molecular analysis of the anthocyanin pigmentation pathway in rice Sorting of storage mRNAs on distinct endoplasmic reticulum membranes in developing rice endosperms |
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