The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Revealing the genetic architecture of FHB resistance in hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) by QTL meta-analysis

Fusarium head blight (FHB) in wheat results in reduced yield and quality and in accumulation of mycotoxins. The objective of this study was to identify genomic regions in wheat involved in the control of FHB resistance applying a QTL meta-analysis approach by combining QTL of 30 mapping populations to propose independent meta-QTL (MQTL). A consensus map was created on which initial QTL were projected. Nineteen MQTL comprising 2–13 initial QTL with widely varying confidence intervals were found on 12 chromosomes. Some of them coincided with genomic regions previously identified (e.g. chromosomes 3BS, 6B), however, some MQTL were newly detected by this study. Separate analysis of populations with the same resistant parent showed a rather high consistency for the Chinese spring wheat donor ‘Sumai 3’, but little consistency for the Chinese donor ‘Wangshuibai’ and the Swiss donor ‘Arina’. According to our results breeders can in future (1) choose parents for crossing not comprising the same resistance loci or QTL intervals, (2) exploit new MQTL, and (3) select markers of some of these MQTL to be used in marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Cloning and characterization of a bile salt hydrolase (<Emphasis Type="Italic">bsh</Emphasis>) from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis>

A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P_bsh) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.     

Molecular genetic mapping of a high-lysine mutant gene (<Emphasis Type="Italic">opaque-16</Emphasis>) and the double recessive effect with <Emphasis Type="Italic">opaque-2</Emphasis> in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertsons Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F_2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F₃ seeds from the F₂ plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F₂ plant marker genotypes and the lysine content of F₃ seeds originating from the two F_2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F_2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F₃ o2o2o16o16 families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F₃ o2o2 and o16o16 families, respectively. The lysine content of seven F₃ families among nine F₃ double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.     

A single-base deletion mutation in <Emphasis Type="Italic">SlIAA9</Emphasis> gene causes tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) <Emphasis Type="Italic">entire</Emphasis> mutant

The entire (e) locus of tomato (Solanum lycopersicum L.) controls leaf morphology. Dominant E and recessive e allele of the locus produce pinnate compound and complex reduced leaves. Previous research had indicated that SlIAA9, an Aux/IAA gene, was involved in tomato leaf morphology. Down-regulation of SlIAA9 gene by antisense transgenic method decreased the leaf complex of tomato and converted tomato compound leaves to simple leaves. The leaf morphology of these transgenic lines was similar with leaf morphology of tomato entire mutant. In this paper, we report that a single-base deletion mutation in the coding region of SlIAA9 gene results in tomato entire mutant phenotypes.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Cloning,expression and characterization of a lipase gene (<Emphasis Type="Italic">lip3</Emphasis>) from<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> LST-03

A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0–35°C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.Communicated by H. Ikeda     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

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.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.