Chromosomal Location of xa19, a Broad-Spectrum Rice Bacterial Blight Resistant Gene from XM5, a Mutant Line from IR24.

Bacterial blight is an important rice disease caused by bacteria named Xanthomonas oryzae pv. oryzae (Xoo). XM5 is an Xoo resistant mutant line with the genetic background of IR24, an Indica Xoo susceptible cultivar, induced by a chemical mutagen N-methyl-N-nitrosourea (MNU). XM5 carries a recessive Xoo resistant gene, xa19. Trisomic analysis was conducted using the cross between XM5 and the trisomic series under the genetic background of IR24, showing that xa19 was located on chromosome 7. The approximate chromosomal location was found using 37 surely resistant plants in the F2 population from XM5 × Kinmaze, which was susceptible to most Japanese Xoo races. The IAS44 line carries a Japonica cultivar Asominori chromosomal segment covering the xa19 locus under the IR24 genetic background. Linkage analysis using the F2 population from the cross between XM5 and IAS44 revealed that xa19 was located within the 0.8 cM region between RM8262 and RM6728. xa19 is not allelic to the known Xoo resistant genes. However, its location suggests that it might be allelic to a lesion-mimic mutant gene spl5, some alleles of which are resistant to several Xoo races. Together with xa20 and xa42, three Xoo resistant genes were induced from IR24 by MNU. The significance of chemical mutagen as a source of Xoo resistance was discussed.

QTL Analysis Revealed One Major Genetic Factor Inhibiting Lesion Elongation by Bacterial Blight (Xanthomonas oryzae pv. oryzae) from a japonica Cultivar Koshihikari in Rice.

Xanthomonas oryzae pv. oryzae (Xoo) is a pathogen that has ravaged the rice industry as the causal agent of bacterial blight (BB) diseases in rice. Koshihikari (KO), an elite japonica cultivar, and ARC7013 (AR), an indica cultivar, are both susceptible to Xoo. Their phenotypic characteristics reveal that KO has shorter lesion length than that of AR. The F2 population from KO × AR results in continuous distribution of lesion length by inoculation of an Xoo race (T7147). Consequently, quantitative trait loci (QTL) mapping of the F2 population is conducted, covering 12 chromosomes with 107 simple sequence repeat (SSR) and insertion/deletion (InDel) genetic markers. Three QTLs are identified on chromosomes 2, 5, and 10. Of them, qXAR5 has the strongest resistance variance effect of 20.5%, whereas qXAR2 and qXAR10 have minor QTL effects on resistance variance, with 3.9% and 2.3%, respectively, for a total resistance variance of 26.7%. The QTLs we examine for this study differ from the loci of BB resistance genes from earlier studies. Our results can help to facilitate understanding of genetic and morphological fundamentals for use in rice breeding programs that are more durable against evolving Xoo pathogens and uncertain climatic temperature.

New Hybrid Spikelet Sterility Gene Found in Interspecific Cross between Oryza sativa and O. meridionalis.

Various kinds of reproductive barriers have been reported in intraspecific and interspecific crosses between the AA genome Oryza species, to which Asian rice (O. sativa) and African rice (O. glaberrima) belong. A hybrid seed sterility phenomenon was found in the progeny of the cross between O. sativa and O. meridionalis, which is found in Northern Australia and Indonesia and has diverged from the other AA genome species. This phenomenon could be explained by an egg-killer model. Linkage analysis using DNA markers showed that the causal gene was located on the distal end of chromosome 1. Because no known egg-killer gene was located in that chromosomal region, this gene was named HYBRID SPIKELET STERILITY 57 (abbreviated form, S57). In heterozygotes, the eggs carrying the sativa allele are killed, causing semi-sterility. This killer system works incompletely: some eggs carrying the sativa allele survive and can be fertilized. The distribution of alleles in wild populations of O. meridionalis was discussed from the perspective of genetic differentiation of populations.

Linkage analysis of rice bacterial blight resistance gene xa20 in XM6, a mutant line from IR24.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is an important disease constraining rice (Oryza sativa L.) production worldwide. The XM6 line was induced by N-methyl-N-nitrosourea from IR24, an Indica cultivar that is susceptible to Philippine and Japanese Xoo races. XM6 was confirmed to carry a recessive gene named xa20, resistant to six Philippine and five Japanese Xoo races. The chromosomal gene location was found using 10 plants with the shortest lesion length in an F2 population consisting of 298 plants from a susceptible Japonica variety Koshihikari × XM6. Analysis using PCR-based DNA markers covering the whole rice genome indicated the gene as located on the distal region of the long arm of chromosome 3. The IKC3 line carries IR24 genetic background with Koshihikari fragment on chromosome 3 where a resistance gene was thought to be located. The F2 population from IKC3 × XM6 clearly showed a bimodal distribution separating resistant and susceptible plants. Further linkage analysis conducted using this F2 population revealed that xa20 is located within the 0.8 cM region flanked by DNA markers KIC3-33.88 (33.0 Mb) and KIC3-34.06 (33.2 Mb). This study yields important findings for resistance breeding and for the genetic mechanism of Xoo resistance.

Phylogenetic analysis of the Si7PPO gene in foxtail millet, Setaria italica, provides further evidence for multiple origins of the negative phenol color reaction phenotype.

To elucidate the diversity and evolution of the Si7PPO gene that controls phenol color reaction (Phr) in foxtail millet, Setaria italica, we analyzed sequence polymorphisms of the Si7PPO gene in 39 accessions consisting of foxtail millet landraces (32 accessions) and their wild ancestor ssp. viridis (seven accessions) collected from various regions in Europe and Asia. The accessions included wild type (positive Phr) and three different types of loss-of-function phenotype (negative Phr), "stop codon type", "TE1-insertion type" and "6-bp duplication type", found in our previous study. We constructed a phylogenetic tree of the gene and found that accessions with positive Phr showed higher genetic diversity at the nucleotide sequence level. We also found that the three different loss-of-function types formed different clusters, suggesting that landraces with negative Phr have multiple origins from three different lineages including both landrace and ssp. viridis accessions with positive Phr.

Molecular and Morphological Divergence of Australian Wild Rice.

Two types of perennial wild rice, Australian Oryza rufipogon and a new taxon Jpn2 have been observed in Australia in addition to the annual species Oryza meridionalis. Jpn2 is distinct owing to its larger spikelet size but shares O. meridionalis-like morphological features including a high density of bristle cells on the awn surface. All the morphological traits resemble O. meridionalis except for the larger spikelet size. Because Jpn2 has distinct cytoplasmic genomes, including the chloroplast (cp), cp insertion/deletion/simple sequence repeats were designed to establish marker systems to distinguish wild rice in Australia in different natural populations. It was shown that the new taxon is distinct from Asian O. rufipogon but instead resembles O. meridionalis. In addition, higher diversity was detected in north-eastern Australia. Reproductive barriers among species and Jpn2 tested by cross-hybridization suggested a unique biological relationship of Jpn2 with other species. Insertions of retrotransposable elements in the Jpn2 genome were extracted from raw reads generated using next-generation sequencing. Jpn2 tended to share insertions with other O. meridionalis accessions and with Australian O. rufipogon accessions in particular cases, but not Asian O. rufipogon except for two insertions. One insertion was restricted to Jpn2 in Australia and shared with some O. rufipogon in Thailand.

Detailed investigation of methylmercury accumulation in rice grain from Hg2+-spiked non-contaminated paddy field soils.

Total-Hg (T-Hg) and methylmercury (MeHg) concentrations in rice grains were measured to understand the MeHg accumulation process. Rice plants were cultivated in Hg2+-spiked non-contaminated soils in experimental pots at three different places. Although soil MeHg concentrations in the pots changed significantly and individually during the rice-growing season, T-Hg concentration of brown rice grain was high at high soil MeHg concentration. In addition, there was no significant variation in T-Hg concentration in brown rice grains from individual panicles or among panicles obtained from the same pot, although the period of growth for each panicle was different. The highest T-Hg concentration of brown rice grains recorded for a panicle was 1.4 ± 0.1 mg kg-1 (n = 8), and the corresponding MeHg ratio was 76%. In addition, the T-Hg and MeHg concentrations in various parts of the brown rice grain-white rice (endosperm), bran, and embryo-were measured. Among the parts of the brown rice grain, the embryo had the highest Hg concentration. Furthermore, Hg concentration in the grain was constant during grain filling. These findings suggest that MeHg formed in soil accumulates in the rice plant during growth and is supplied to the rice grains continuously for the entire duration of the grain development period.

HWA1- and HWA2-Mediated Hybrid Weakness in Rice Involves Cell Death, Reactive Oxygen Species Accumulation, and Disease Resistance-Related Gene Upregulation.

Hybrid weakness is a type of reproductive isolation in which F1 hybrids of normal parents exhibit weaker growth characteristics than their parents. F1 hybrid of the Oryza sativa Indian cultivars 'P.T.B.7' and 'A.D.T.14' exhibits hybrid weakness that is associated with the HWA1 and HWA2 loci. Accordingly, the aim of the present study was to analyze the hybrid weakness phenotype of the 'P.T.B.7' × 'A.D.T.14' hybrids. The height and tiller number of the F1 hybrid were lower than those of either parent, and F1 hybrid also exhibited leaf yellowing that was not observed in either parent. In addition, the present study demonstrates that SPAD values, an index correlated with chlorophyll content, are effective for evaluating the progression of hybrid weakness that is associated with the HWA1 and HWA2 loci because it accurately reflects degree of leaf yellowing. Both cell death and H2O2, a reactive oxygen species, were detected in the yellowing leaves of the F1 hybrid. Furthermore, disease resistance-related genes were upregulated in the yellowing leaves of the F1 hybrids, whereas photosynthesis-related genes tended to be downregulated. These results suggest that the hybrid weakness associated with the HWA1 and HWA2 loci involves hypersensitive response-like mechanisms.

Segregation Distortion Observed in the Progeny of Crosses Between Oryza sativa and O. meridionalis Caused by Abortion During Seed Development.

Wild rice relatives having the same AA genome as domesticated rice (Oryza sativa) comprise the primary gene pool for rice genetic improvement. Among them, O. meridionalis and O. rufipogon are found in the northern part of Australia. Three Australian wild rice strains, Jpn1 (O. rufipogon), Jpn2, and W1297 (O. meridionalis), and one cultivated rice cultivar Taichung 65 (T65) were used in this study. A recurrent backcrossing strategy was adopted to produce chromosomal segment substitution lines (CSSLs) carrying chromosomal segments from wild relatives and used for trait evaluation and genetic analysis. The segregation of the DNA marker RM136 locus on chromosome 6 was found to be highly distorted, and a recessive lethal gene causing abortion at the seed developmental stage was shown to be located between two DNA markers, KGC6_10.09 and KGC6_22.19 on chromosome 6 of W1297. We name this gene as SEED DEVELOPMENT 1 (gene symbol: SDV1). O. sativa is thought to share the functional dominant allele Sdv1-s (s for sativa), and O. meridionalis is thought to share the recessive abortive allele sdv1-m (m for meridionalis). Though carrying the sdv1-m allele, the O. meridionalis accessions can self-fertilize and bear seeds. We speculate that the SDV1 gene may have been duplicated before the divergence between O. meridionalis and the other AA genome Oryza species, and that O. meridionalis has lost the function of the SDV1 gene and has kept the function of another putative gene named SDV2.

High-resolution mapping and characterization of xa42, a resistance gene against multiple Xanthomonas oryzae pv. oryzae races in rice (Oryza sativa L.).

Improvement of resistance against rice bacterial blight (BB) disease is an important breeding strategy in breeding programs across the world, especially in Africa and southern Asia where BB is more prevalent. This report describes a high-resolution map and characterization of xa42 at XA42 locus, a rice BB resistance gene in XM14, a mutant line originating from IR24. The candidate gene region was narrowed down from 582 kb, which had been obtained in our previous study, to 57 kb. XM14 shows brown spots in its leaves like lesion mimic mutants. This line also shows a shorter stature than the original cultivar IR24. In XA42 gene segregating populations, homozygotes of xa42 allele were consistently resistant to the six Japanese Xanthomonas oryzae pv. oryzae races used for this study. They also showed brown spots and markedly short stature compared with the other genotypes, suggesting that xa42 gene exhibits pleiotropic effects.

Identification and linkage analysis of a new rice bacterial blight resistance gene from XM14, a mutant line from IR24.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a chief factor limiting rice productivity worldwide. XM14, a rice mutant line resistant to Xoo, has been obtained by treating IR24, which is susceptible to six Philippine Xoo races and six Japanese Xoo races, with N-methyl-N-nitrosourea. XM14 showed resistance to six Japanese Xoo races. The F2 population from XM14 × IR24 clearly showed 1 resistant : 3 susceptible segregation, suggesting control of resistance by a recessive gene. The approximate chromosomal location of the resistance gene was determined using 10 plants with shortest lesion length in the F2 population from XM14 × Koshihikari, which is susceptible to Japanese Xoo races. DNA marker-assisted analysis revealed that the gene was located on chromosome 3. IAS16 line carries IR24 genetic background with a Japonica cultivar Asominori segment of chromosome 3, on which the resistance gene locus was thought to be located. The F2 population from IAS16 × XM14 showed a discrete distribution. Linkage analysis indicated that the gene is located around the centromeric region. The resistance gene in XM14 was a new gene, named XA42. This gene is expected to be useful for resistance breeding programs and for genetic analysis of Xoo resistance.

Distribution of Hwc2-1, a causal gene of a hybrid weakness, in the World Rice Core collection and the Japanese Rice mini Core collection: its implications for varietal differentiation and artificial selection.

A pair of complementary genes, Hwc1-1 at HWC1 locus and Hwc2-1 at HWC2 locus, cause a weakness phenomenon in rice. For this study, we performed haplotype analysis around the HWC2 locus in two core collections comprising 119 accessions. We also examined reactions to phenol and Xanthomonas oryzae pv. oryzae (Xoo) Japanese race I. To elucidate the genetic relations among all accessions, we analyzed their banding patterns of 40 Indel markers covering the rice genome. The classification by Indel markers was almost consistent with that using 4,357 SNPs. The testcross with Hwc1-1 carrier indicated that 37 accessions carried Hwc2-1 allele, whereas 82 carried hwc2-2 allele. Strong association between HWC2 and Ph genes was observed. Based on 14 DNA markers around HWC2 locus and Ph genotype, the 119 accessions were divided into 50 haplotypes. To examine the HWC2 candidate chromosomal region specifically, the 'haplotype group' characterized by the six DNA markers closely linked with HWC2 were analyzed. Hwc2-1 carriers had the same haplotype group. Some hwc2-2 haplotype groups were associated with resistance against the Xoo race. The relation between varietal differentiation and haplotypes around the HWC2 locus was discussed, along with its breeding significance.

Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at frequencies of ca. 25-67 % and those with negative Phr were broadly found in Europe and Asia. The stop codon type was found in 285 accessions and was broadly distributed in Europe and Asia, whereas the TE-1 insertion type was found in 99 accessions from Europe and Asia but was not found in India. The 6-bp duplication type was found in only 8 accessions from Nansei Islands (Okinawa Prefecture) of Japan. We also analyzed Phr in the wild ancestor and concluded that the negative Phr type was likely to have originated after domestication of foxtail millet. It was also implied that negative Phr of foxtail millet arose by multiple independent loss of function of PPO gene through dispersal because of some advantages under some environmental conditions and human selection as in rice and barley.

Genetic analysis of ion-beam induced extremely late heading mutants in rice.

Two extremely late heading mutants were induced by ion beam irradiation in rice cultivar 'Taichung 65': KGM26 and KGM27. The F2 populations from the cross between the two mutants and Taichung 65 showed clear 3 early: 1 late segregation, suggesting control of late heading by a recessive gene. The genes identified in KGM26 and KGM27 were respectively designated as FLT1 and FLT2. The two genes were mapped using the crosses between the two mutants and an Indica cultivar 'Kasalath'. FLT1 was located on the distal end of the short arm of chromosome 8. FLT2 was located around the centromere of chromosome 9. FLT1 might share the same locus as EHD3 because their chromosomal location is overlapping. FLT2 is inferred to be a new gene because no gene with a comparable effect to that of this gene was mapped near the centromere of chromosome 9. In crosses with Kasalath, homozygotes of late heading mutant genes showed a large variation of days to heading, suggesting that other genes affected late heading mutant genes.

Molecular relationships between Australian annual wild rice, Oryza meridionalis, and two related perennial forms.

BACKGROUND: The perennial, Oryza rufipogon distributed from Asia to Australia and the annual O. meridionalis indigenous to Australia are AA genome species in the Oryza. However, recent research has demonstrated that the Australian AA genome perennial populations have maternal genomes more closely related to those of O. meridionalis than to those found in Asian populations of O. rufipogon suggesting that the Australian perennials may represent a new distinct gene pool for rice. RESULTS: Analysis of an Oryza core collection covering AA genome species from Asia to Oceania revealed that some Oceania perennials had organellar genomes closely related to that of O meridionalis (meridionalis-type). O. rufipogon accessions from New Guinea carried either the meridionalis-type or rufirpogon-type (like O. rufipogon) organellar genomes. Australian perennials carried only the meridionalis-type organellar genomes when accompanied by the rufipogon-type nuclear genome. New accessions were collected to better characterize the Australian perennials, and their life histories (annual or perennial) were confirmed by field observations. All of the material collected carried only meridionalis-type organellar genomes. However, there were two distinct perennial groups. One of them carried an rufipogon-type nuclear genome similar to the Australian O. rufipogon in the core collection and the other carried an meridionalis-type nuclear genome not represented in the existing collection. Morphologically the rufipogon-type shared similarity with Asian O. rufipogon. The meridionalis-type showed some similarities to O. meridionalis such as the short anthers usually characteristic of annual populations. However, the meridionalis-type perennial was readily distinguished from O. meridionalis by the presence of a larger lemma and higher number of spikelets. CONCLUSION: Analysis of current accessions clearly indicated that there are two distinct types of Australian perennials. Both of them differed genetically from Asian O. rufipogon. One lineage is closely related to O. meridionalis and another to Asian O. rufipogon. The first was presumed to have evolved by divergence from O. meridionalis becoming differentiated as a perennial species in Australia indicating that it represents a new gene pool. The second, apparently derived from Asian O. rufipogon, possibly arrived in Australia later.

Chromosomal Location of HCA1 and HCA2, Hybrid Chlorosis Genes in Rice.

MANY POSTZYGOTIC REPRODUCTIVE BARRIER FORMS HAVE BEEN REPORTED IN PLANTS: hybrid weakness, hybrid necrosis, and hybrid chlorosis. In this study, linkage analysis of the genes causing hybrid chlorosis in F(2) generation in rice, HCA1 and HCA2, was performed. HCA1 and HCA2 are located respectively on the distal regions of the short arms of chromosomes 12 and 11. These regions are known to be highly conserved as a duplicated chromosomal segment. The molecular mechanism causing F(2) chlorosis deduced from the location of the two genes was discussed. The possibility of the introgression of the chromosomal segments encompassing HCA1 and/or HCA2 was also discussed from the viewpoint of Indica-Japonica differentiation.

Fine mapping and allelic dosage effect of Hwc1, a complementary hybrid weakness gene in rice.

Hybrid weakness is a reproductive barrier that is found in many plant species. In rice, the hybrid weakness caused by two complementary genes, Hwc1 and Hwc2, has been surveyed intensively. However, their gene products and the molecular mechanism that causes hybrid weakness have remained unknown. We performed linkage analyses of Hwc1, narrowed down the area of interest to 60 kb, and identified eight candidate genes. In the F(2) population, in which both Hwc1 and Hwc2 genes were segregated, plants were separable into four classes according to their respective phenotypes: severe type, semi-severe type, F(1) type, and normal type. Severe type plants show such severe symptoms that they could produce only tiny shoot-like structures; they were unable to generate roots. Genetic analyses using closely linked DNA markers of the two genes showed that the symptoms of the F(2) plants were explainable by the genotypes of Hwc1 and Hwc2. Weakness was observed in plants that have both Hwc1 and Hwc2. In Hwc1 homozygote, the symptoms worsened and severe type or semi-severe type plants appeared. Consequently, Hwc1 should have a gene dosage effect and be a semi-dominant gene. The dosage effect of Hwc2 was recognizable, but it was not so severe as that in Hwc1. These results are useful to elucidate the mechanism that causes the hybrid weakness phenomenon and the role of each causal gene in hybrid weakness.

Phylogenetic analysis of the rDNA intergenic spacer subrepeats and its implication for the domestication history of foxtail millet, Setaria italica.

We sequenced ribosomal DNA intergenic spacer subrepeats and their flanking regions of foxtail millet landraces from various regions in Europe and Asia and its wild ancestor to elucidate phylogenetic differentiation within each of types I-III found in our previous work and to elucidate relationships among these three types. Type I was classified into seven subtypes designated as Ia-Ig based on subrepeat sequences; C repeats downstream of those subrepeats are also polymorphic. Of these, subtypes Ia-Id and Ig were found in foxtail millet landraces. Subtypes Ia and Ib were distributed broadly throughout Asia and Europe. Subtype Ic was distributed in China, Korea and Japan. Subtype Id has a 20-bp deletion in subrepeat 3 and has a unique C repeat sequence. This subtype was found in a morphologically primitive landrace group from Afghanistan and northwestern Pakistan and differed greatly from other type I subtypes, implying that these landraces were domesticated independently. Subtypes Ig was found in a landrace from Pakistan and Ia and Ie-Ig were in six wild ancestor accessions. Type II was also highly polymorphic and four subtypes were found and designated as subtypes IIa-IId, but sequence analyses indicated type III as monomorphic. The present work indicates that type III should be classified as a subtype of type II (subtype IIe). Sequence polymorphism of subrepeats of types I-III indicated that subrepeats of subtype IIa are greatly divergent from others. Relationships among types I-III are much more complicated than anticipated based on previous RFLP work.

Ribosomal DNA intergenic spacer sequence in foxtail millet, Setaria italica (L.) P. Beauv. and its characterization and application to typing of foxtail millet landraces.

We determined the sequence of ribosomal DNA (rDNA) intergenic spacer (IGS) of foxtail millet isolated in our previous study, and identified subrepeats in the polymorphic region. We also developed a PCR-based method for identifying rDNA types based on sequence information and assessed 153 accessions of foxtail millet. Results were congruent with our previous works. This study provides new findings regarding the geographical distribution of rDNA variants. This new method facilitates analyses of numerous foxtail millet accessions. It is helpful for typing of foxtail millet germplasms and elucidating the evolution of this millet.