Isolation and sequence analysis of wheat NBS-LRR type disease resistance gene analogs using degenerate PCR primers.

Isolation of disease resistance gene analogs (RGAs) using the conserved motifs of the resistance genes has attracted considerable attention since it was first reported more than a decade ago. In this study, RGAs are isolated using homology-based PCR to target the nucleotide binding site (NBS) conserved regions from hexaploid wheat varieties and a few accessions of wild types. Based on sequence similarity analysis, 83 of the sequenced clones were clustered as groups. Of these RGAs, 40 were in the NBS-LLR class, containing kinase-1a (GGVGKTT or GGVGKTA), kinase-2 (KRFLIVLDDXW), kinase-3a (GSXIVVITTR or GCXVLATTR), and the GLPL motif of the NBS-spanning region. Among these, 15 contained possible intron regions, similar to Avena sativa O2 NBS-LLR type disease resistance gene (AF078874), and one to Rpm1 of rice and Yr10 and Lr10 of wheat. To our knowledge, this is the first observation of an intronic site within the P-loop domain of wheat RGAs. We detected an unspecified motif (VMVCVS) between the kinase-1a and kinase-2 domains within our clones. Additionally, one of the clones showed replacement with the kinase-3a motif with an undefined sequence.

Genetic relationships among perennial and annual Cicer species growing in Turkey assessed by AFLP fingerprinting.

AFLP markers were used to assess genetic relationships among Cicer species with distribution in Turkey. Genetic distances were computed among 47 Cicer accessions representing four perennial and six annual species including chickpea, using 306 positions on AFLP gels. AFLP-based grouping of species revealed two clusters, one of which includes three perennial species, Cicer montbretii, Cicer isauricum and Cicer anatolicum, while the other cluster consists of two subclusters, one including one perennial, Cicer incisum, along with three annuals from the second crossability group ( Cicer pinnatifidum, Cicer judaicum and Cicer bijugum) and the other one comprising three annuals from the first crossability group ( Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Consistent with previous relationship studies in the same accession set using allozyme and RAPD markers, in AFLP-based relationships, C. incisum was the closest perennial to nearly all annuals, and C. reticulatum was the closest wild species to C. arietinum. Cluster analysis revealed the grouping of all accessions into their distinct species-clusters except for C. reticulatum accessions, ILWC247, ILWC242 and TR54961; the former was found to be closer to the C. arietinum accessions while the latter two clustered with the C. echinospermum group. Small genetic distance values were detected among C. reticulatum accessions (0.282) and between C. reticulatum and C. arietinum (0.301) indicating a close genetic similarity between these two species. Overall, the AFLP-based genetic relationships among accessions and species were congruous with our previous study of genetic relationships using allozymes. The computed level of AFLP variation and its distribution into within and between Cicer species paralleled the previous report based on RAPD analyses. AFLP analysis also confirmed the presence of the closest wild relatives and previous projections of the origin of chickpea in southern Turkey. Results presented in this report indicate that AFLP analysis is an efficient and reliable marker technology in determination of genetic variation and relationships in the genus Cicer. Obviously, the use of AFLP fingerprinting in constructing a detailed genetic map of chickpea and cloning, and characterizing economically important traits would be promising as well.

RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment.

No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be betaalphabeta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus species. A fragment of the gene (ldh), coding for approximately 72% of the lactate dehydrogenase enzyme from Rhizopus oryzae, was amplified using degenerate primers by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the amplified fragment containing betaalphabeta nucleotide binding site, substrate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme productions in industrial scales.

The use of microsatellite DNA markers for soybean genotype identification.

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) n /(TA) n and (ATT) n /(TAA) n that are composed of tandemly repeated 2-5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, "n", results in PCR product length differences. The SSR alleles present at three (AT) n /(TA) n and four (ATT) n /(TAA) n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.

Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea (Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

Length polymorphisms of simple sequence repeat DNA in soybean.

The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.

Light regulation of protein synthesis factor EF-G in pea chloroplasts.

The activity of pea chloroplast elongation factor G (EF-G), a nuclear-coded protein required for the elongation cycle of chloroplast protein synthesis, is regulated in response to light. In pea seedlings germinated and grown under continuous white or red light, EF-G specific activity reaches a maximum between days 10 to 15, and then decreases. EF-G activity is almost undetectable in extracts from dark-grown seedlings. When 13-day dark-grown pea seedlings are transferred to light, EF-G specific activity reaches a higher value after 2 to 3 days than observed in seedlings grown under continuous light. The small and large subunits of ribulose bisphosphate carboxylase continue to accumulate after EF-G specific activity has reached maximum levels. Cytoplasmically synthesized components of the chloroplast protein synthetic apparatus, such as EF-G, may help coordinate cytoplasmic and nuclear events with chloroplast gene expression during light-induced chloroplast differentiation.