Biomedical subjects

N K Blake

Publications and source records attributed to N K Blake.

6 recordsLinked to original sources

PubMed2004

Genome-specific primer sets for starch biosynthesis genes in wheat.

Common wheat (Triticum aestivum L.,2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat-rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence.Exon-anchored primers, which amplify across introns,allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT),and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (Ssl). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms(SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of theseSNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.

Base Sequence↗

PubMed2002

Multiple origins of allopolyploid Aegilops triuncialis.

Polyploidization is a key component of plant evolution. The number of independent origins of polyploid species traditionally has been underestimated. The objective of this study was to ascertain the number of origins of a tetraploid Aegilops species. We screened 84 primer sets to identify genome-specific primer sets for the tetraploid wheat relative [Aegilops triuncialis (UUCC genome)] and its diploid progenitors [Ae. umbellulata (UU genome) and Ae. caudata (CC genome)]. Primer sets G12 and G43 were U genome-specific and D21 was a C genome-specific primer. DNA sequence comparison of the G43 locus was used to estimate the number of polyploidization events in the formation of Ae. triuncialis. Parsimony analysis of G43 data revealed at least two independent formations of Ae. triuncialis. In the chloroplast hotspot region, located between genes rbcL and petA, sequence analysis suggested that at least three polyploidization origins might have occurred independently. Ae. triuncialis appears to be a tetraploid derived from multiple origins with minimal genome change after its formation.

Base Sequence↗

PubMed2000

Properties of sequence-tagged-site primer sets influencing repeatability.

The polymerase chain reaction (PCR) has become a standard procedure in plant genetics, and is the basis for many emerging genomics approaches to mapping and gene identification. One advantage of PCR is that sequence information for primer sets can be exchanged between laboratories, obviating the need for exchange and maintenance of biological materials. Repeatability of primer sets, whereby the same products are amplified in different laboratories using the same primer set, is important to successful exchange and utilization. We have developed several hundred sequence-tagged site (STS) primer sets for wheat and barley. The ability of the primer sets to generate reproducible amplifications in other laboratories has been variable. We wished to empirically determine the properties of the primer sets that most influenced repeatability. A total of 96 primer sets were tested with four genomic DNA samples on each of four thermocyclers. All major bands were repeatable across all four thermocylers for approximately 50% of the primer sets. Characteristics most often associated with differences in repeatability included primer GC content and 3'-end stability of the primers. The propensity for primer-dimer formation was not a factor in repeatability. Our results provide empirical direction for the development of repeatable primer sets.

DNA Primers↗

PubMed1999

Phylogenetic reconstruction based on low copy DNA sequence data in an allopolyploid: the B genome of wheat.

Study of bread wheat (Triticum aestivum) may help to resolve several questions related to polyploid evolution. One such question regards the possibility that the component genomes of polyploids may themselves be polyphyletic, resulting from hybridization and introgression among different polyploid species sharing a single genome. We used the B genome of wheat as a model system to test hypotheses that bear on the monophyly or polyphyly of the individual constituent genomes. By using aneuploid wheat stocks, combined with PCR-based cloning strategies, we cloned and sequenced two single-copy-DNA sequences from each of the seven chromosomes of the wheat B genome and the homologous sequences from representatives of the five diploid species in section Sitopsis previously suggested as sister groups to the B genome. Phylogenetic comparisons of sequence data suggested that the B genome of wheat underwent a genetic bottleneck and has diverged from the diploid B genome donor. The extent of genetic diversity among the Sitopsis diploids and the failure of any of the Sitopsis species to group with the wheat B genome indicated that these species have also diverged from the ancestral B genome donor. Our results support monophyly of the wheat B genome.

Chromosome Mapping↗

PubMed1995

Variability in wheat based on low-copy DNA sequence comparisons.

The chromosomes of the B genome of hexaploid wheat (AABBDD) do not pair completely with those of any of the diploid species with genomes similar to B. Various biochemical and molecular analyses have suggested that each of the five diploid species in section Sitopsis of Triticum are ancestral to B. These observations have led to the hypothesis that the B genome may be polyphyletic, descending from more than one diploid ancestor. This hypothesis may account for differences between the wheat B genome and the diploids and also for variability that currently exists among different wheat accessions. In this study, we cloned and compared nucleotide sequences for three low-copy DNA fragments from the B and D genomes of several wheat accessions and from diploid relatives of the B and D genomes. Our results suggested that the amount of DNA sequence variability in wheat is low, although somewhat more variability existed in the B genome than in the D genome. The B genome of wheat was significantly diverged from all the Sitopsis diploid species, and Triticum speltoides was closer to B than to other members of this section. The D genome of wheat was very similar to that of its progenitor, Triticum tauschii. No evidence for a polyphyletic origin of the B genome was found. A more parsimonious hypothesis is that the wheat B genome diverged from its diploid ancestor after the original hybridization event occurred.

Cloning, Molecular↗

PubMed1987

The isolation of fumB mutants of Escherichia coli.

Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35.7) is deleted. Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium. These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93.4). A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.

Autoradiography↗