A 2600-locus chromosome bin map of wheat homoeologous group 2 reveals interstitial gene-rich islands and colinearity with rice.

The complex hexaploid wheat genome offers many challenges for genomics research. Expressed sequence tags facilitate the analysis of gene-coding regions and provide a rich source of molecular markers for mapping and comparison with model organisms. The objectives of this study were to construct a high-density EST chromosome bin map of wheat homoeologous group 2 chromosomes to determine the distribution of ESTs, construct a consensus map of group 2 ESTs, investigate synteny, examine patterns of duplication, and assess the colinearity with rice of ESTs assigned to the group 2 consensus bin map. A total of 2600 loci generated from 1110 ESTs were mapped to group 2 chromosomes by Southern hybridization onto wheat aneuploid chromosome and deletion stocks. A consensus map was constructed of 552 ESTs mapping to more than one group 2 chromosome. Regions of high gene density in distal bins and low gene density in proximal bins were found. Two interstitial gene-rich islands flanked by relatively gene-poor regions on both the short and long arms and having good synteny with rice were discovered. The map locations of two ESTs indicated the possible presence of a small pericentric inversion on chromosome 2B. Wheat chromosome group 2 was shown to share syntenous blocks with rice chromosomes 4 and 7.

A 2500-locus bin map of wheat homoeologous group 5 provides insights on gene distribution and colinearity with rice.

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

Analysis of expressed sequence tag loci on wheat chromosome group 4.

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat.

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.

Interaction of Chlamydomonas dynein with tubulin.

Studies were conducted to determine if dynein could bind to unpolymerized tubulin. Tubulin alone normally fractionated in the included volume of a molecular sieve Bio-Gel A-1.5m column. Incubated together, tubulin and dynein coeluted in the void volumn, suggesting that a complex had formed between the two. In addition, immunoelectron microscopy revealed preassembled microtubules were labeled with biotin antibody only when incubated in both dynein and biotinylated tubulin, evidence that dynein with bound biotinylated tubulin had decorated the microtubules. A fraction of the tubulin could be dissociated from dynein by addition of ATP and vanadate, as assayed by molecular sieve chromatography followed by densitometry of gels, suggesting that some tubulin bound to the B end of the dynein arm. Additional tubulin dissociated from the dynein under conditions of high salt. These studies, together with those indicating that tubulin blocked the A end of the dynein arm from binding to microtubules and promoted the interaction of two arms at their A ends, provide evidence that the A end of the arm also can bind tubulin. Thus, the tubulin subunits, themselves, on a microtubule rather than a particular surface lattice structure formed by adjacent protofilaments may provide the binding sites for both ends of the dynein arm.

Microtubule crossbridging by chlamydomonas dynein.

Dynein, obtained from axonemes of Chlamydomonas, binds by both its A and B ends to microtubules assembled from twice cycled (2X) and purified (6S) brain tubulin as well as to microtubules in native spindles, thereby inducing microtubule crossbridging. The two ends of the dynein arm exhibit distinct binding characteristics for the different microtubule preparations. Greater than 99% of the dynein arms are bound exclusively by their B ends to microtubules assembled from 6S tubulin in the presence of dynein and decorated to saturation. In contrast, greater than 80% of the dynein arms are bound by both their A and B ends to and, therefore, crossbridge 6S microtubules that are only partially dynein decorated. Binding of the A end of the dynein arm to saturated 6S microtubules can be enhanced by destabilizing the binding of the B end upon addition of ATP and vanadate. These observations suggest that Chlamydomonas dynein arms can bind by their A ends to microtubules assembled from 6S tubulin only when the B ends of the arms either are not bound or are bound but do not occupy all available dynein binding sites. Dynein exhibits a slight preference for binding by its A end to microtubules assembled from 2X tubulin and containing microtubule associated proteins (MAPs). Approximately 90% of the dynein arms crossbridge adjacent 2X microtubules that are only partially decorated. But as saturation of these microtubules with dynein is approached, the majority of the arms are bound solely by their A ends, while a smaller percentage are bound by their B ends or by both their A and B ends. These studies indicate that the type of microtubule as well as the degree of saturation of the microtubule with dynein can determine whether microtubule crossbridging occurs.

A view of plant dehydrins using antibodies specific to the carboxy terminal peptide.

Dehydrins are characterized by the consensus KIKEKLPG amino acid sequence found near the carboxy terminus, and usually repeated from one to many times within the protein. A synthetic peptide containing this consensus sequence was used to produce specific antibodies that recognize dehydrins in a wide range of plants. This range covered two families of monocots, viz. Gramineae (Hordeum vulgare L., Triticum aestivum L., Zea mays L., Oryza sativa L.) and Liliaceae (Allium sativa L.), and five families of dicots, Malvaceae (Gossypium hirsutum L.), Solanaceae (Lycopersicon esculentum L.), Brassicaceae (Raphanus sativus L.), Fabaceae (Vigna unguiculata L.), and Cucurbitaceae (Cucumis sativus L.). Two families of gymnosperms, Pinaceae (Pinus edulis Engelm.) and Ginkgoaceae (Ginkgo biloba L.), were also included. For several plants in which dehydrin cDNA and genomic clones have previously been characterized, it now appears that the dehydrin family of proteins is larger, and the regulation of dehydrin expression much more complex, than earlier studies have shown.