PCR-based markers for the powdery mildew resistance gene<Emphasis Type="Italic"> Pm4a</Emphasis> in wheat

Gene tagging is the basis of marker-assisted selection and map-based cloning. To develop PCR-based markers for Pm4a, a dominant powdery mildew resistance gene of wheat, we surveyed 46 group 2 microsatellite markers between Pm4a near-isogenic line (NIL) CI 14124 and the recurrent parent Chancellor (Cc). One of the markers, gwm356, detected polymorphism and was used for genotyping an F₂ population of 85 plants derived from CI 14124 × Cc. Linkage mapping indicated that Xgwm356 was linked to Pm4a at a distance of 4.8 cM. To identify more PCR-based markers for Pm4a, we also converted the restriction fragment length polymorphism marker BCD1231 linked to it into a sequence-tagged site (STS) marker. The STS primer designed based on the end sequences of BCD1231 amplified an approximately 1.6-kb monomorphic band in both parents. Following digestion of the products with the four-cutter enzymes HaeIII and MspI, it was discovered that the band from CI 14124 consisted of at least two products, one of which had a digestion pattern different from the band from Cc. In the F₂ population, the cleaved polymorphism revealed by the STS marker between the parents co-segregated with powdery mildew resistance. To design Pm4a-specific PCR markers, the 1.6-kb band from Cc and the fragment associated with Pm4a in CI 14124 were sequenced and compared. Based on these sequences a new PCR marker was identified, which detected a 470-bp product only in the Pm4a-containing lines. These PCR-based markers provide a cost-saving option for marker-assisted selection of Pm4a.Communicated by F. Salamini     

RFLP markers linked to powdery mildew resistance genes Pm1, Pm2, Pm3, and Pm4 in wheat.

Near-isogenic lines (NILs) and their recurrent parent Chancellor (Cc) were used to identify restriction fragment length polymorphic markers linked to powdery mildew (Blumeria graminis (DC.) E.O. Speer f.sp. tritici) resistance genes Pm1, Pm2, Pm3, and Pm4 in wheat (Triticum aestivum L. em. Thell). By mapping these polymorphic markers in F2 progenies from crosses of the NILs with Cc, it was found that Pm1 cosegregated with a polymorphic locus detected by DNA probe CDO347; Pm2 was linked to a locus detected by probe BCD1871 with a distance of 3.5 cM; Pm3b was linked to a locus detected by probe BCD1434 with a distance of 1.3 cM; Pm4a cosegregated with Xbcd1231-2A(2) and Xcdo678-2A, and was closely flanked by Xbcd1231-2A(1) and Xbcd292-2A both with a distance of 1.5 cM. Aneuploid mapping of these markers indicated that locus Xcdo347-7A is on 7AL, Xbcd1871-5D on 5DS, Xbcd1434-1A on 1AS, and loci Xbcd292-2A and Xcdo678-2A are on 2AL. The same polymorphic fragments detected in the Pm3b NIL by Xbcd1434-1A were found in Pm3a NIL using several enzyme digestions.     

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

高效产氢菌B49菌株adh和L-ldh基因克隆及序列分析

设计细菌adh基因和L-ldh基因简并引物,采用降落PCR并结合Cassette PCR方法从高效产氢菌B49中扩增全长基因.共获得两段基因组DNA片段.其中一段序列长1902 bp.包括adh基因开放阅读框 1101 bp,共编码366个氨基酸,编码产物分子量为 39.71 kD,理论等电点为pH5.93.该基因与Clostridium thermocellum ATCC 27405的adh位点序列一致性为73%.另一段序列长2490bp,包括L-ldh基因开放阅读框951 bp,共编码316个氨基酸,编码产物分子量为34.23 kD,理论等电点为pH 6.09.该基因与Bacillus megaterium的L-ldh位点一致性为74%.试验结果表明,采用CodeHop和Genefisher程序化设计的简并引物可信性强,阳性率高,CodeHop设计简并引物效果要好于Genefisher.adh和L-ldh的成功克隆为通过代谢工程手段敲除adh和L-ldh基因提供了科学依据,从而为进一步提高B49产氢能力的代谢工程研究奠定基础.     

高效产氢菌B49菌株adh和L-ldh基因克隆及序列分析

设计细菌adh基因和L-ldh基因简并引物, 采用降落PCR并结合Cassette PCR方法从高效产氢菌B49中扩增全长基因。共获得两段基因组DNA片段。其中一段序列长1902 bp, 包括adh基因开放阅读框1101 bp, 共编码366个氨基酸, 编码产物分子量为39.71 kD, 理论等电点为pH 5.93。该基因与Clostridium thermocellum ATCC 27405的adh位点序列一致性为73%。另一段序列长2490 bp, 包括L-ldh基因开放阅读框951 bp, 共编码316个氨基酸, 编码产物分子量为34.23 kD, 理论等电点为pH 6.09。该基因与Bacillus megaterium的L-ldh位点一致性为74%。试验结果表明, 采用CodeHop和Genefisher程序化设计的简并引物可信性强, 阳性率高, CodeHop设计简并引物效果要好于Genefisher。adh和L-ldh的成功克隆为通过代谢工程手段敲除adh和L-ldh基因提供了科学依据, 从而为进一步提高B49产氢能力的代谢工程研究奠定基础。     

Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans.

The positively acting regulator gene QUTA from Aspergillus nidulans has been identified and located within a cluster of quinic acid utilisation (QUT) genes isolated within a recombinant phage lambda (lambda Q1). The DNA sequence of the QUTA gene reveals a single uninterrupted reading frame coding for a protein of mw 90.416 Kd. The QUTA protein sequence has a protein motif in the form of a putative "DNA finger" that shows strong homology to other such motifs in the GAL4, PPR1, ARGRII, LAC9 and QA1F regulatory gene products of S. cerevisiae, K. lactis and N. crassa. The data presented confirm the view deduced by genetical analysis that the QUTA gene of A. nidulans encodes a protein capable of interacting with QUT specific DNA sequences.     

High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria

Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.     

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.