Genetic and physical mapping of AvrPi7, a novel avirulence gene of Magnaporthe oryzae using physical position-ready markers

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating crop diseases worldwide. The avirulence gene corresponding to rice blast resistance gene Pi7 in field isolate CHL346 was inherited as a single gene, designated AvrPi7, in a segregating population consisting of 189 ascospore progenies derived from a cross between field isolates CHL346 and CHL42. In order to determine the chromosomal location of the AvrPi7 locus, a total of 121 simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of reference isolate 70-15 of M. oryzae. Linkage analysis of the locus with these SSR markers showed that eight SSR markers on chromosome 1 were linked to the locus, among which the closest flanking markers MS1-9 and MS1-15 were 3.2 and 16.4 cM from the locus, respectively. For fine mapping, additional PCR-based makers including eight SSR markers and three candidate avirulence gene (CAG) markers were developed in the region flanking both markers. The AvrPi7 locus was genetically delimited within a 1.6-cM region flanked by markers MS1-21 and MS1-22, and co-segregated with the marker CAG2. To construct a physical map of the AvrPi7 locus, molecular markers linked to the Avr gene were mapped on the supercontigs of the ref-erence isolate 70-15 through bioinformation analysis (BIA). Consequently, the AvrPi7 locus was delim-ited to a 75-kb interval flanked by markers MS1-21 and MS1-22 based on the reference sequence. Merodiploids observed in this study are also discussed.     

Fine mapping of a semidwarf gene sd-g in indica rice（Oryza sativa L.）

The semidwarf gene sd-g which has been usedin indiea rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived fromthe cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region.The sd-g was further mapped between two microsatellitemarkers SSR5-1 and SSR5-51, with genetic distances of 0.1and 0.3 cM, respectively, while cosegregated with SSR418. ABAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloningof the sd-g gene.     

Fine mapping of the awn gene on chromosome 4 in rice by association and linkage analyses

Awnness is a key trait in rice domestication, yet no studies have been conducted on fine mapping or association mapping of the rice awn gene. In this study, we investigated the awnness and genotype of a core collection of 303 cultivated rice varieties and a BC5F2 segregating population of 200 individuals. Combining association and linkage analyses, we mapped the awnness related genes to chromosome 4. Primary association analysis using 24 SSR markers revealed five loci significantly associated with awnness on chromosome 4. The associated markers cover previously identified regions. Fine association mapping was conducted using another 29 markers within a 4-Mb region, covering the associated marker in34, which is close to the awn gene Awn4.1. Seven associated markers were revealed, distributed over an 870-kb region. Combining the fine association mapping and linkage analysis of awnness in the 200 BC5F2 segregating population, we finally identified a 330-kb region as the candidate region for Awn4.1. The results indicate that combining association mapping and linkage mapping provides an efficient and precise approach to both genome-wide mapping and fine mapping of rice genes.     

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 （presenescing leaf） mutant was obtained from a japonica variety Zhonghua 11 via radiation of ^60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the biggest length, while the leaves of the wild variety could keep green for 25--35 d. In this study, genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene （psl1） located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nipponbare and 93-11. The psl1 was further mapped between two STS markers, STS2-19 and STS2-26, with genetic distances of 0.43 and 0.11 cM, respectively, while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus, the region being delimited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Characterization and mapping of a white panicle mutant gene in rice

A spontaneous white panicle mutant was found from the F6 progenies of an indicajaponica cross.The mutant exhibits white stripes on its basal leaves while the panicles,rachis and pedicel are milky white colored at flowering stage.Genetic analysis in an F2 population from the cross of Zhi7/white panicle mutant indicates that the white panicle phenotype is controlled by a single recessive nuclear gene,tentatively termed as wp(t).Using microsatellite markers,the wp(t) gene was anchored between the markers of SSR101 and SSR63.9 with a map distance of 2.3 and 0.8cM,respectively,and co-segregated with the marker of SSR17 on rice chromosome 1.     

Genetic analysis and molecular mapping of a presenescing leaf gene psl1 in rice (Oryza sativa L.)

A rice psl1 (presenescing leaf) mutant was obtained from a japonica variety Zhonghua 11 via radiation of 60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the big-gest length,while the leaves of the wild variety could keep green for 25―35 d. In this study,genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene (psl1) located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nippon-bare and 93-11. The psl1 was further mapped be-tween two STS markers,STS2-19 and STS2-26,with genetic distances of 0.43 and 0.11 cM,respectively,while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus,the region being delim-ited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

Over-expression of an Arabi-dopsisd δ-OAT gene enhances salt and drought tolerance in transgenic rice

δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsis δ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCI concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenie rice. Furthermore, the over-expression of δ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%--41% more than that of control line sunder 0.1 mol/L NaCI stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Genetic analysis and mapping of rice （Oryza sativa L.） male-sterile （OsMS-L） mutant

A rice male-sterile mutant OsMS-L of japonica cultivar 9522 background, was obtained in M4 population treated with ^60Co γ-Ray. Genetic analysis indicated that the male.sterile phenotype was controlled by a single recessive gene. Results of tissue section showed that at microspore stage, OsMS-L tapetum was retarded. Then tapetal calls expanded and microspores degenerated. No matured pollens were observed in OsMS-L anther locus. To map OsMS-L locus, an F2 population was constructed from the cross between the OsMS-L (japonica) and LongTeFu B(indica). Firstly, the OsMS-L locus was roughly mapped between two SSR markers, RM109 and RM7562 on chromosome 2. And then eleven polymorphic markers were developed for further fine fine-mapping. At last the OsMS-L locus was mapped between the two lnDel markers, Lhsl0 and Lhs6 with genetic distance of 0.4 cM, respectively. The region was delimited to 133 kb. All these results were useful for further cloning and functional analysis of OsMS-L.     

dr1127： A novel gene of Deinococcus radiodurans responsible for oxidative stress

The functional analysis of dr1127,a novel gene in Deinococcus radiodurans was performed in this pa-per. The dr1127 gene was found occasionally in our microarray and 2-DE gel experiments. Mutation of the dr1127 gene decreased the γ-radiation and H2O2 resistance of D. radiodurans,and weakened the scavenging abilities of cell extracts for free radicals (superoxide anion,hydrogen peroxide,and hy-droxyl radical). Further oxidative damage assays demonstrated that the purified DR1127 protein of D. radiodurans could bind to double stranded DNA in vitro and protect DNA from oxidative damage in this way. These results suggest that the dr1127 gene is an important gene that can maintain γ-radiation and oxidative resistance in D. radiodurans and may take part in the oxidative stress process.     

Identification of <Emphasis Type="Italic">Fallopia</Emphasis> species based on the chloroplast <Emphasis Type="Italic">mat</Emphasis>K gene sequences

In order to develop an efficient identification method for Fallopia plants and drugs, molecular analysis of the partial matK gene sequences was performed on 6 Fallopia species. Based on the matK sequences, a phylogenetic tree was constructed, which showed that different populations of inter- and intra-species could be specified and distinguished. The matK gene sequences of the 6 Fallopia species were all found to be of 1 271 bp in length, with some nucleotide variations throughout the entire sequences. The nucleotide difference at position 1 041 could distinguish F. denticulata from others, while specific nucleotide at position 1 154 became identification markers for F. aubertii. Moreover, four specific marker sites for F. multiflora var. ciliinerve at positions 216, 224, 1 060 and 1 179, seven for F. convolvulus at 318, 765, 772, 874, 936, 952 and 1 036, and seven for F. dentate alata at 111, 192, 366, 450, 457, 1 032 and 1 074 were also observed. By detecting the marker nucleotides and analyzing the phylogenetic relationship, the botanical origins of five inspected drugs were determined, suggesting that matK sequences can be used for authenticating Fallopia plants and drugs.     

Genetic analysis and gene fine mapping of a rolling leaf mutant (<Emphasis Type="Italic">rl</Emphasis><Subscript><Emphasis Type="Italic">11(t)</Emphasis></Subscript>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The exploration of new genes controlling rice leaf shape is an important foundation for rice functional genomics and plant archi-tecture improvement. In the present study, we identified a rolling leaf mutant from indica variety Yuefeng B, named rl_11(t), which exhibited reduced plant height, rolling and narrow leaves. Leaves in rl_11(t) mutant showed abnormal number and morphology of veins compared with those in wild type plants. In addition, rl_11(t) mutant was less sensitive to the inhibitory effect of auxin than the wild type. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Gene Rl_11(t) was initially mapped between SSR markers RM6089 and RM124 on chromosome 4. Thirty-two new STS markers around the Rl_11(t) region were developed for fine mapping. A physical map encompassing the Rl_11(t) locus was constructed and the target gene was finally delimited to a 31.6 kb window between STS4-25 and STS4-26 on BAC AL606645. This provides useful information for cloning of Rl_11(t) gene.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

Transpositional behaviour of the Ds element in the Ac/Ds system in rice

Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F1 to F5 gen- erations indicated that the frequencies of germinal transposition increased over successive genera- tions and reached 54.2% in F3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking se- quences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.     

B-Valued Dyadic Derivative

The principles of the new maximal operator H＊ we defined are discussed. We prove that it is bounded from martingale Hardy-Lorentz L^Xp.q[0,1） to the Lorentz L^Xp.q[0,1） for 1/2〈 p〈∞, 0〈~ q ≤ ∞, where X is any Banach space. When the Banach space X has the RN property, the sequence dnHnf converges to f a.e. Meanwhile the convergence in L^Xp norm for 1≤p〈∞ is a consequence of that the family functions K （n∈N） is an approximate identity.     

Transfer of a eubacteria-type cell division site-determining factor CrldfnD 8ene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, MesosUgma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer （LGT） of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coil inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.     

Synthesis,characterization and photoluminescence of aluminum <Emphasis Type="Italic">N</Emphasis>-aryloxo functionalized <Emphasis Type="Italic">β</Emphasis>-ketoiminate complexes

The synthesis, characterization and luminescent properties of aluminum complexes containing a dianionic N-aryloxo functionalized β-ketoiminate ligand are presented. 4-(2-Hydroxy-5-R-phenyl)imino-2-pentanone (R = Me, L¹H₂; R = tert-butyl, L²H₂) ligands reacted with AlEt₃ in tetrahydrofuran to give the aluminum complexes (L¹AlEt)₂ (1) and (L²AlEt)₂ (2) in reasonable isolated yields. X-ray diffraction revealed that complexes 1 and 2 have solvent-free centrosymmetric dimeric structures, and each aluminum center has distorted trigonal bipyramidal geometry. At room temperature, complexes 1 and 2 exhibit blue photoluminescence in acetonitrile with maximum emission wavelengths of 419 and 413 nm, respectively.     

The R- and AR-indices： Complementing the h-index

Based on the foundation laid by the h-index we introduce and study the R- and AR-indices. These new indices eliminate some of the disadvantages of the h-index, especially when they are used in combina-tion with the h-index. The R-index measures the h-core’s citation intensity, while AR goes one step further and takes the age of publications into account. This allows for an index that can actually in-crease and decrease over time. We propose the pair (h, AR) as a meaningful indicator for research evaluation. We further prove a relation characterizing the h-index in the power law model.