Identification and analysis of QTLs controlling cold tolerance at the reproductive stage and validation of effective QTLs in cold-tolerant genotypes of rice (Oryza sativa L.).

Low temperature or cold stress is one of the major constraints of rice production and productivity in temperate rice-growing countries and high-altitude areas in the tropics. Even though low temperature affects the rice plant in all stages of growth, the percent seed set is damaged severely by cold and this reduces the yield potential of cultivars significantly. In this study, a new source of cold-tolerant line, IR66160-121-4-4-2, was used as a donor parent with a cold-sensitive cultivar, Geumobyeo, to produce 153 F(8) recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis. QTL analysis with 175 polymorphic simple sequence repeat (SSR) markers and composite interval mapping identified three main-effect QTLs (qPSST-3, qPSST-7, and qPSST-9) on chromosomes 3, 7, and 9. The SSR markers RM569, RM1377, and RM24545 were linked to the identified QTLs for cold tolerance with respect to percent seed set using cold-water (18-19 degrees C) irrigation in the field and controlled air temperature (17 degrees C) in the greenhouse. The total phenotypic variation for cold tolerance contributed by the three QTLs was 27.4%. RILs with high percent seed set under cold stress were validated with linked DNA markers and by haplotype analysis that revealed the contribution of progenitor genomes from the tropical japonica cultivar Jimbrug (Javanica) and temperate japonica cultivar Shen-Nung89-366. Three QTLs contributed by the cold-tolerant parent were identified which showed additive effect on percent seed set under cold treatment. This study demonstrated the utility of a new phenotyping method as well as the identification of SSR markers associated with QTLs for selection of cold-tolerant genotypes to improve temperate rice production.

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta.

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F(2) population derived from a cross between an introgression line, 'IR71033-121-15', from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, 'Junambyeo'. Resistance to BPH (biotype 1) was evaluated using 190 F(3) families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

The pi40 gene for durable resistance to rice blast and molecular analysis of pi40-advanced backcross breeding lines.

Rice blast severely reduces production in both irrigated and water-stressed upland ecosystems of tropical and temperate countries. Nearly 50 blast resistance genes have been identified and some of those are incorporated into several rice cultivars. However, most of the resistance genes break down in a few years because of their race specificity and the rapid change in pathogenicity of the blast fungus (Magnaporthe grisea). The objective of this study was to analyze advanced backcross breeding lines (ABL) possessing the gene Pi40 for durable rice blast resistance. In all, 4 resistant genotypes, 4 japonica cultivars, and 10 monogenic differential rice genotypes with some known resistance genes were bioassayed in the greenhouse using seven sequential plantings and 29 virulent M. grisea isolates of Korea. The genotypes with the Pi40 gene had <3% diseased leaf area, which was significantly below the disease threshold level of 40% considered for durable blast resistance. Moreover, the genotypes with the Pi40 gene expressed compatibility with only two to three virulent M. grisea isolates supporting durability of resistance, in contrast to susceptible cultivars with >50% diseased leaf area and 10 compatible isolates. Of the 10 known resistance genes tested, Piz-t, Piz-5, and Pi9 showed differential reactions to the pathogen isolates in seven plantings. Genotyping of the ABL with 260 simple sequence repeat (SSR) markers revealed rapid conversion toward recurrent parent genotypes with fewer donor chromosomal segments (5.3 to 14.5%). Our study based on a sequential testing and background selection of breeding lines with the resistance gene Pi40 provided valuable information for durable blast resistance breeding in rice.

A novel gene, Pi40(t), linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice.

Rice blast disease caused by Magnaporthe grisea is a continuous threat to stable rice production worldwide. In a modernized agricultural system, the development of varieties with broad-spectrum and durable resistance to blast disease is essential for increased rice production and sustainability. In this study, a new gene is identified in the introgression line IR65482-4-136-2-2 that has inherited the resistance gene from an EE genome wild Oryza species, O. australiensis (Acc. 100882). Genetic and molecular analysis localized a major resistance gene, Pi40(t), on the short arm of chromosome 6, where four blast resistance genes (Piz, Piz-5, Piz-t, and Pi9) were also identified, flanked by the markers S2539 and RM3330. Through e-Landing, 14 BAC/PAC clones within the 1.81-Mb equivalent virtual contig were identified on Rice Pseudomolecule3. Highly stringent primer sets designed for 6 NBS-LRR motifs located within PAC clone P0649C11 facilitated high-resolution mapping of the new resistance gene, Pi40(t). Following association analysis and detailed haplotyping approaches, a DNA marker, 9871.T7E2b, was identified to be linked to the Pi40(t) gene at the 70 Kb chromosomal region, and differentiated the Pi40(t) gene from the LTH monogenic differential lines possessing genes Piz, Piz-5, Piz-t, and Pi-9. Pi40(t) was validated using the most virulent isolates of Korea as well as the Philippines, suggesting a broad spectrum for the resistance gene. Marker-assisted selection (MAS) and pathotyping of BC progenies having two japonica cultivar genetic backgrounds further supported the potential of the resistance gene in rice breeding. Our study based on new gene identification strategies provides insight into novel genetic resources for blast resistance as well as future studies on cloning and functional analysis of a blast resistance gene useful for rice improvement.

High-resolution mapping of a new brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in rice (Oryza sativa L.).

Brown planthopper (BPH) is a destructive insect pest of rice in Asia. Identification and the incorporation of new BPH resistance genes into modern rice cultivars are important breeding strategies to control the damage caused by new biotypes of BPH. In this study, a major resistance gene, Bph18(t), has been identified in an introgression line (IR65482-7-216-1-2) that has inherited the gene from the wild species Oryza australiensis. Genetic analysis revealed the dominant nature of the Bph18(t) gene and identified it as non-allelic to another gene, Bph10 that was earlier introgressed from O. australiensis. After linkage analysis using MapMaker followed by single-locus ANOVA on quantitatively expressed resistance levels of the progenies from an F2 mapping population identified with marker allele types, the Bph18(t) gene was initially located on the subterminal region of the long arm of chromosome 12 flanked by the SSR marker RM463 and the STS marker S15552. The corresponding physical region was identified in the Nipponbare genome pseudomolecule 3 through electronic chromosome landing (e-landing), in which 15 BAC clones covered 1.612 Mb. Eleven DNA markers tagging the BAC clones were used to construct a high-resolution genetic map of the target region. The Bph18(t) locus was further localized within a 0.843-Mb physical interval that includes three BAC clones between the markers R10289S and RM6869 by means of single-locus ANOVA of resistance levels of mapping population and marker-gene association analysis on 86 susceptible F2 progenies based on six time-point phenotyping. Using gene annotation information of TIGR, a putative resistance gene was identified in the BAC clone OSJNBa0028L05 and the sequence information was used to generate STS marker 7312.T4A. The marker allele of 1,078 bp completely co-segregated with the BPH resistance phenotype. STS marker 7312.T4A was validated using BC2F2 progenies derived from two temperate japonica backgrounds. Some 97 resistant BC2F2 individuals out of 433 screened completely co-segregated with the resistance-specific marker allele (1,078 bp) in either homozygous or heterozygous state. This further confirmed a major gene-controlled resistance to the BPH biotype of Korea. Identification of Bph18(t) enlarges the BPH resistance gene pool to help develop improved rice cultivars, and the PCR marker (7312.T4A) for the Bph18(t) gene should be readily applicable for marker-assisted selection (MAS).

Dynamics of Xanthomonas oryzae pv. oryzae Populations in Korea and Their Relationship to Known Bacterial Blight Resistance Genes.

ABSTRACT Developing resistant cultivars requires an understanding of the dynamics of the pathogen populations as well as the genetics of host resistance. Bacterial leaf blight (BB), caused by the vascular pathogen Xanthomonas oryzae pv. oryzae, has become one of the most devastating diseases of rice. We demonstrate here the quantitative analyses of responses of near-isogenic lines carrying various BB resistance (R) genes and R-gene combinations against 16 X. oryzae pv. oryzae isolates representing Korean BB pathotypes. The estimated main effects of each R gene against the 16 isolates identified prominent differences in BB pathotypes between Korea and other countries. Three major aspects of our quantitative observations and statistical analysis are (i) strong and broad resistance of xa5; (ii) independent and additive genetic actions of Xa4, xa5, and Xa21 under digenic or trigenic status; and (iii) a strong quantitative complementation effect contributed by the functional alleles of Xa4 and Xa21. We conclude that the pyramid line containing genes Xa4, xa5, and Xa21 would be the most promising and valuable genotype for improving Korean japonica cultivars for BB resistance.

DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice.

Bacterial leaf blight caused by the bacterial pathogen Xanthomonas oryzae pv oryzae (Xoo) limits rice yield in all major rice-growing regions of the world, especially in irrigated lowland and rainfed conditions where predisposition factors favor disease development to epidemic proportions. Since bacterial pathogens are difficult to manage, development of host plant resistance is the most effective means of disease management. As many as 24 major genes conferring resistance to various races of the pathogen have been identified and utilized in rice breeding programs. However, large-scale and long-term cultivation of varieties carrying a single gene for resistance resulted in a significant shift in pathogen race frequency with consequent breakdown of resistance in these cultivars. To combat the problem of resistance breakdown, pyramiding of resistance genes into different cultivars is being carried out. Pyramiding of resistance genes is now possible with molecular markers that are developed for individual genes. This review discusses the various bacterial blight resistance genes identified and their corresponding molecular markers developed for breeding durable resistance into modern rice cultivars.

Comparative RFLP mapping of a wild rice, Oryza officinalis, and cultivated rice, O. sativa.

A comparative RFLP map was constructed in a wild rice, Oryza officinalis, by using 139 genomic and cDNA probes that had been used previously to map RFLPs in O. sativa. Nine of the 12 chromosomes of O. officinalis were highly homosequential to those of O. sativa. A major rearrangement of gene order was detected in chromosome 1 and small inversions were found in chromosomes 3 and 11. Fourteen translocated RFLP markers were found, and chromosome 11 contained a high frequency of such translocated segments. Results were consistent with meiotic and trisomic analysis, which suggested that the genomes of O. officinalis and O. sativa were similar. Applications of comparative maps in plant breeding and gene cloning are discussed.

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F1 hybrids (AE) was 2.29 to 4.85 with a range of 0-8 bivalents. F1 hybrids were completely male sterile. We did not obtain any BC1 progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F1 hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC1 plants varied from 28 to 31, and all were male sterile. BC2 plants had 24-28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC2 F2 progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC2 progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC2 F4 progenies 4 were resistant to BPH and 1 to race 6 of BB. F3 segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.

RFLP analysis of rice (Oryza sativa L.) introgression lines.

Fifty-two introgression lines (BC2F8) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.

Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice, O. sativa L.

Sterile AC hybrids between cultivated Oryza sativa (AA) and a distant wild species, O. officinalis (CC), were backcross to O. sativa. Most of the BC1 progenies were allotriploid (AAC), a few were hypotriploid. AAC progenies were again backcrossed to O. sativa. BC2 progenies consisting of disomic or aneuploid individuals were examined for the presence of O. officinalis traits. Eleven different traits from O. officinalis were identified in these progenies. Segregation data in the subsequent generations suggest that these traits are monogenic in nature. Two of these genes - for resistance to BPH and WBPH - are of value in rice improvement. The extremely low recovery of recombinant progenies is in agreement with the very low amount of pairing between A and C genomes. Because of this restricted recombination, the genotype of the recurrent parent was reconstituted after two backcrosses only. Thus, the BC2 progenies look remarkably similar to O. sativa. Most of them are stable and fertile and also interfertile with other O. sativa breeding lines. Some of the BPH-and WBPH-resistant progenies are comparable in yield to the best O. sativa parents and are being evaluated as varietal possibilities.