Biomedical subjects

L E Talbert

Publications and source records attributed to L E Talbert.

10 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↗

PubMed2001

Identification of barley genome segments introgressed into wheat using PCR markers.

Barley has several important traits that might be used in the genetic improvement of wheat. For this report, we have produced wheat-barley recombinants involving barley chromosomes 4 (4H) and 7 (5H). Wheat-barley disomic addition lines were crossed with 'Chinese Spring' wheat carrying the phlb mutation to promote homoeologous pairing. Selection was performed using polymerase chain reaction (PCR) markers to identify lines with the barley chromosome in the ph1b background. These lines were self pollinated, and recombinants were identified using sequence-tagged-site (STS) primer sets that allowed differentiation between barley and wheat chromosomes. Several recombinant lines were isolated that involved different STS-PCR markers. Recombination was confirmed by allowing the lines to self pollinate and rescreening the progeny via STS-PCR. Progeny testing confirmed 9 recombinants involving barley chromosome 4 (4H) and 11 recombinants involving barley chromosome 7 (5H). Some recombinants were observed cytologically to eliminate the possibility of broken chromosomes. Since transmission of the recombinant chromosomes was lower than expected and since seed set was reduced in recombinant lines, the utility of producing recombinants with this method is uncertain.

DNA, Plant↗

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

Molecular analysis of evolutionary patterns in U genome wild wheats.

The theory of pivotal-differential evolution states that one genome of polyploid wheats remains stable (i.e., pivotal) during evolution, while the other genome or genomes may become modified (i.e., differential). A proposed mechanism for apparent modification of the differential genome is that different polyploid species with only one genome in common may exchange genetic material. In this study, we analyzed a set of sympatric and allopatric accessions of tetraploid wheats with the genomic constitutions UM and UC. The U genome of these species is from Triticum umbellulatum and is considered to be the pivotal genome. The M and C genomes, from T. comosum and T. dichasians, respectively, are considered to be the differential genomes. Low copy DNA was analyzed using "sequence tagged site" primer sets in the polymerase chain reaction, followed by digestion with restriction enzymes. Genetic similarity matrices based on shared restriction fragments showed that sympatric accessions of different U genome tetraploid species did not tend to share more restriction fragments than did allopatric accessions. Thus, no evidence for introgression was found. Analysis of the diploid progenitor species showed that the U genome was less variable than the M and C genomes. Additionally, comparison of diploid and polyploid species using genome-specific primer sets suggests a possible polyphyletic origin for T. triunciale and T. machrochaetum. Thus, our results suggest that the differential nature of the M and C genomes may be the result of variability introduced by the diploid progenitors and not the result of frequent introgression events after formation of the polyploid.

Base Sequence↗

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↗

PubMed1989

Mu transposable elements are structurally diverse and distributed throughout the genus Zea.

The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of the Mu family of transposable elements. All characterized Mu elements contain similar approximately 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20-100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to the Mu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously cloned Mu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termed Mu4 and Mu5, have an organization characteristic of transposable elements and possess approximately 200-bp Mu terminal inverted repeats that flank internal DNA, which is unrelated to other cloned Mu elements. Mu4 and Mu5 are both flanked by 9-bp direct repeats as has been observed for other Mu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions of Mu4 and Mu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally from Mu-similar termini. The distribution of these elements in maize lines and related species suggests that Mu elements are an ancient component of the maize genome. Moreover, the structure of the Mu termini and the fact that Mu termini are found flanking different internal sequences leads us to speculate that Mu termini once may have been capable of transposing as independent entities.

Base Sequence↗

PubMed1988

Sequence, genomic distribution and DNA modification of a Mu1 element from non-mutator maize stocks.

The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.

Base Sequence↗

PubMed1988

Characterization of a highly conserved sequence related to mutator transposable elements in maize.

Mutator stocks of maize exhibit a high mutation rate correlated with the activity of a family of transposable elements. Mu1 and, to a lesser extent, the closely related Mu1.7 elements are responsible for most mutator-induced mutations that have been characterized. These elements are found in 10-60 copies in mutator stocks, and zero to a few intact elements exist in nonmutator maize stocks. Additionally, the component parts of Mu elements exist separately in the maize genome. The Mu terminal inverted repeats are found in multiple copies in all maize lines and related Zea species tested, and Mu internal sequences exist unassociated with Mu termini. In the present paper, we describe the structure and genomic distribution of one Mu-homologous sequence termed MRS-A (for Mu-related sequence). DNA sequencing shows that MRS-A is closely related to the internal region of Mu1 and Mu1.7 elements. However, it has no Mu termini and does not have the structure of a transposable element. This sequence is present in one or two copies in all maize lines and is highly conserved in the genus Zea. A similar sequence exists in a species within the genus most closely related to Zea, Tripsacum dactyloides, although the T. dactyloides genome does not contain any Mu termini or intact Mu elements. Furthermore, an RNA transcript homologous to MRS-A and its flanking DNA is found in both mutator and nonmutator maize plants. These results suggest that MRS-A represents a stable, functional region of the maize genome, and we speculate that a similar sequence was encompassed by Mu termini to generate a Mu transposable element.

Base Sequence↗