Localization of <Emphasis Type="Italic">pms3</Emphasis>, a gene for photoperiod-sensitive genic male sterility,to a 28.4-kb DNA fragment

Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F₂ population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3.     

Fine mapping and candidate gene analysis of <Emphasis Type="Italic">hwh1</Emphasis> and <Emphasis Type="Italic">hwh2</Emphasis>, a set of complementary genes controlling hybrid breakdown in rice

Hybrid breakdown (HB), a phenomenon of reduced viability or fertility accompanied with retarded growth in hybrid progenies, often arises in the offspring of intersubspecific hybrids between indica and japonica in rice. We detected HB plants in F₈ recombinant inbred lines derived from the cross between an indica variety, Milyang 23, and a japonica variety, Tong 88-7. HB plants showed retarded growth, with fewer tillers and spikelets. Genetic analysis revealed that HB was controlled by the complementary action of two recessive genes, hwh1 and hwh2, originating from each of both parents, which were fine-mapped on the short arm of chromosome 2 and on the near centromere region of the long arm of chromosome 11, respectively. A comparison of the sequences of candidate genes among both parents and HB plants revealed that hwh1 encoded a putative glucose-methanol-choline oxidoreductase with one amino acid change compared to Hwh1 and that hwh2 probably encoded a putative hexose transporter with a six amino acid insertion compared to Hwh2. Investigation of the distribution of these alleles among 54 japonica and indica cultivars using candidate gene-based markers suggested that the two loci might be involved in developing reproductive barriers between two subspecies.     

Molecular characterization the <Emphasis Type="Italic">YABBY</Emphasis> gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis> and expression analysis of <Emphasis Type="Italic">OsYABBY1</Emphasis>

Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.     

Efficient authentic fine mapping of the rice blast resistance gene <Emphasis Type="Italic">Pik-h</Emphasis> in the <Emphasis Type="Italic">Pik</Emphasis> cluster,using new <Emphasis Type="Italic">Pik-h</Emphasis>-differentiating isolates

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Characterization and identification of the candidate gene of rice thermo-sensitive genic male sterile gene <Emphasis Type="Italic">tms5</Emphasis> by mapping

Previous research has demonstrated that the thermo-sensitive genic male-sterile (TGMS) gene in rice was regulated by temperature. TGMS rice is important to hybrid rice production because the application of the TGMS system in two-line breeding is cost-effective, simple, efficient and overcomes the limitations of the cytoplasmic male sterility (CMS) system. AnnongS is the first discovered and deeply studied TGMS rice line in China. Previous studies have suggested that AnnongS-1 and Y58S, two derivative TGMS lines of AnnongS, were both controlled by a single recessive gene named tms5, which was genetically mapped on chromosome 2. In the current study, three populations (AnnongS-1 × Nanjing11, Y58S × Q611, and Y58S × Guanghui122) were developed to investigate the tms5 gene molecular map. Analysis of recombination events of sterile samples, utilizing 125 probes covering the tms5 region, suggested that the tms5 gene was physically mapped to a 19 kb DNA fragment between two markers, 4039-1 and 4039-2, located on the BAC clone AP004039. Following the construction of a physical map between the two markers, ONAC023, a member of the NAC (NAM-ATAF-CUC-related) gene family, was identified as the candidate of the tms5 gene.     

Molecular dissection of <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedrovirus <Emphasis Type="Italic">orf8</Emphasis> gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac16 interacts with baculoviral and cellular proteins. Bm8/Ac16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bm8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.      

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Fine mapping of the rice thermo-sensitive genic male-sterile gene <Emphasis Type="Italic">tms5</Emphasis>

AnnongS-1, a thermo-sensitive genic male-sterile (TGMS) rice line, has a new TGMS gene. Genetic analysis indicated that the sterility of AnnongS-1 was controlled by a single resessive gene named tms5. In our previous studies based on an F₂ population from the cross between AnnongS-1 and Nanjing11, tms5 was mapped on chromosome 2. Recently, a RIL (recombinant inbred line) population from the same cross was developed and used for the fine mapping of the tms5 gene. Molecular marker techniques combined with BSA (bulked segregant analysis) were used. As a result, two AFLP markers (AF10, AF8), one RAPD marker (RA4), one STS marker (C365-1), one CAPs marker (G227-1) and four SSR markers (RM279, RM492, RM327, RM324) were found to be closely linked to tms5 gene. The DNA sequences of the RFLP marker of C365 and G227 were found in GenBank, and on the basis of these sequences, many primers were designed to amplify the two parents and their RIL population plants. Finally, the tms5 gene was mapped between STS marker C365-1 and CAPs marker G227-1 at a distance of 1.04 cM from C365-1 and 2.08 cM from G227-1.Communicated by H.F. LinskensY.G. Wang and Q.H. Xing contributed equally to this contribution.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Fine mapping of <Emphasis Type="Italic">S31</Emphasis>, a gene responsible for hybrid embryo-sac abortion in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Partial abortion of female gametes and the resulting semi-sterility of indica × japonica inter-subspecific rice hybrids have been ascribed to an allelic interaction, which can be avoided by the use of wide compatibility varieties. To further understand the genetic mechanism of hybrid sterility, we have constructed two sets of hybrids, using as male parent either the typical japonica variety Asominori, or the wide compatibility variety 02428; and as female, a set of 66 chromosome segment substitution lines in which various chromosomal segments from the indica variety IR24 have been introduced into a common genetic background of Asominori. Spikelet semi-sterility was observed in hybrid between CSSL34 and Asominori, which is known to carry the sterility gene S31 (Zhao et al. in Euphytica 151:331–337, 2006). Cytological analysis revealed that the semi-sterility of the CSSL34 × Asominori hybrid was caused primarily by partial abortion of the embryo sac at the stage of the mitosis of the functional megaspore. A population of 1,630 progeny of the three-way cross (CSSL34 × 02428) × Asominori was developed to map S31. Based on the physical location of linked molecular markers, S31 was thereby delimited to a 54-kb region on rice chromsome 5. This fragment contains eight predicted open reading frames, four of which encode known proteins and four putative proteins. These results are relevant to the map-based cloning of S31, and the development of marker-assisted transfer of non-sterility allele inducing alleles to breeding germplasm, to allow for a more efficient exploitation of heterosis in hybrid rice.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Characterization of a regulatory gene, <Emphasis Type="Italic">aveR</Emphasis>, for the biosynthesis of avermectin in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.     

<Emphasis Type="Italic">HvCEBiP</Emphasis>, a gene homologous to rice chitin receptor <Emphasis Type="Italic">CEBiP</Emphasis>, contributes to basal resistance of barley to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background  

Rice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses.