Genome wide linkage disequilibrium in Chinese asparagus bean (Vigna. unguiculata ssp. sesquipedialis) germplasm: implications for domestication history and genome wide association studies.

Association mapping of important traits of crop plants relies on first understanding the extent and patterns of linkage disequilibrium (LD) in the particular germplasm being investigated. We characterize here the genetic diversity, population structure and genome wide LD patterns in a set of asparagus bean (Vigna. unguiculata ssp. sesquipedialis) germplasm from China. A diverse collection of 99 asparagus bean and normal cowpea accessions were genotyped with 1127 expressed sequence tag-derived single nucleotide polymorphism markers (SNPs). The proportion of polymorphic SNPs across the collection was relatively low (39%), with an average number of SNPs per locus of 1.33. Bayesian population structure analysis indicated two subdivisions within the collection sampled that generally represented the 'standard vegetable' type (subgroup SV) and the 'non-standard vegetable' type (subgroup NSV), respectively. Level of LD (r(2)) was higher and extent of LD persisted longer in subgroup SV than in subgroup NSV, whereas LD decayed rapidly (0-2 cM) in both subgroups. LD decay distance varied among chromosomes, with the longest (≈ 5 cM) five times longer than the shortest (≈ 1 cM). Partitioning of LD variance into within- and between-subgroup components coupled with comparative LD decay analysis suggested that linkage group 5, 7 and 10 may have undergone the most intensive epistatic selection toward traits favorable for vegetable use. This work provides a first population genetic insight into domestication history of asparagus bean and demonstrates the feasibility of mapping complex traits by genome wide association study in asparagus bean using a currently available cowpea SNPs marker platform.

Microspore embryogenesis: assignment of genes to embryo formation and green vs. albino plant production.

Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories "cell rescue, defense, and virulence"; "metabolism"; "transcription"; and "transport". These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.

A branched-chain amino acid aminotransferase gene isolated from Hordeum vulgare is differentially regulated by drought stress.

Differential display was used to isolate cDNA clones showing differential expression in response to ABA, drought and cold in barley seedling shoots. One drought-regulated cDNA clone (DD12) was further analyzed and found to encode a branched-chain amino acid aminotransferase (HvBCAT-1). A genomic clone was isolated by probing the Morex BAC library with the cDNA clone DD12 and the structure of Hvbcat-1 was elucidated. The coding region is interrupted by six introns and contains a predicted mitochondrial transit peptide. Hvbcat1 was mapped to chromosome 4H. A comparison was made to rice and Arabidopsis genes to identify conserved structural patterns. Complementation of a yeast (Saccharomyces cerevisiae) double knockout strain revealed that HvBCAT-1 can function as the mitochondrial (catabolic) BCATs in vivo. Transcript levels of Hvbcat-1, increased in response to drought stress. As the first enzyme in the branched-chain amino acid (BCAA) catabolic pathway, HvBCAT-1 might have a role in the degradation of BCAA. Degradation of BCAA could serve as a detoxification mechanism that maintains the pool of free branched-chain amino acids at low and non toxic levels, under drought stress conditions.

Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.

Barley Dhn13 encodes a KS-type dehydrin with constitutive and stress responsive expression.

Dehydrins (DHNs) compose a family of intrinsically unstructured proteins that have high water solubility and accumulate during late seed development, low temperature or water deficit conditions, and are thought to play a protective role in freezing and drought tolerance in plants. Twelve Dhn genes were previously described in the barley genome. Here, we report an additional member of this multigene family, Dhn13. The Dhn13 gene is located in chromosome 4 near marker MWG634 and encodes a 107-amino acid KS-type DHN. Semi-quantitative reverse transcriptase PCR data indicated that Dhn13 is constitutively expressed in seedling tissues and embryos of developing seeds. Microarray data were consistent with these results and showed a considerable increase of Dhn13 transcripts when plants were subjected to chilling and freezing temperatures. The highest transcript levels where observed in anthers. The presence of ABRE, MYC, DRE, and POLLEN1LELAT52 regulatory elements in the putative Dhn13 promoter region is in agreement with expression data.

Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map.

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5' and 3' sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.

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.

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.

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.

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags.

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.

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).

A chromosome bin map of 2148 expressed sequence tag loci of wheat homoeologous group 7.

The objectives of this study were to develop a high-density chromosome bin map of homoeologous group 7 in hexaploid wheat (Triticum aestivum L.), to identify gene distribution in these chromosomes, and to perform comparative studies of wheat with rice and barley. We mapped 2148 loci from 919 EST clones onto group 7 chromosomes of wheat. In the majority of cases the numbers of loci were significantly lower in the centromeric regions and tended to increase in the distal regions. The level of duplicated loci in this group was 24% with most of these loci being localized toward the distal regions. One hundred nineteen EST probes that hybridized to three fragments and mapped to the three group 7 chromosomes were designated landmark probes and were used to construct a consensus homoeologous group 7 map. An additional 49 probes that mapped to 7AS, 7DS, and the ancestral translocated segment involving 7BS also were designated landmarks. Landmark probe orders and comparative maps of wheat, rice, and barley were produced on the basis of corresponding rice BAC/PAC and genetic markers that mapped on chromosomes 6 and 8 of rice. Identification of landmark ESTs and development of consensus maps may provide a framework of conserved coding regions predating the evolution of wheat genomes.

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.

Lower fitness of hatchery and hybrid rainbow trout compared to naturalized populations in Lake Superior tributaries.

We have documented an early life survival advantage by naturalized populations of anadromous rainbow trout Oncorhynchus mykiss over a more recently introduced hatchery population and outbreeding depression resulting from interbreeding between the two strains. We tested the hypothesis that offspring of naturalized and hatchery trout, and reciprocal hybrid crosses, survive equally from fry to age 1+ in isolated reaches of Lake Superior tributary streams in Minnesota. Over the first summer, offspring of naturalized females had significantly greater survival than offspring of hatchery females in three of four comparisons (two streams and 2 years of stocking). Having an entire naturalized genome, not just a naturalized mother, was important for survival over the first winter. Naturalized offspring outperformed all others in survival to age 1+ and hybrids had reduced, but intermediate, survival relative to the two pure crosses. Averaging over years and streams, survival relative to naturalized offspring was 0.59 for hybrids with naturalized females, 0.37 for the reciprocal hybrids, and 0.21 for hatchery offspring. Our results indicate that naturalized rainbow trout are better adapted to the conditions of Minnesota's tributaries to Lake Superior so that they outperform the hatchery-propagated strain in the same manner that many native populations of salmonids outperform hatchery or transplanted fish. Continued stocking of the hatchery fish may conflict with a management goal of sustaining the naturalized populations.

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.

Liberalized screening for blunt carotid and vertebral artery injuries is justified.

BACKGROUND: Current literature suggests that blunt carotid injuries (BCIs) and vertebral artery injuries (BVIs) are more common than once appreciated. Screening criteria have been suggested, but only one previous study has attempted to identify factors that predict the presence of BCI/BVI. This current study was conducted for two reasons. First, we wanted to determine the incidence of BCI/BVI in our institution. Second, we wanted to determine the incidence of abnormal four-vessel cerebral angiograms ordered for injuries and signs believed to be associated with BCI/BVI and thus to determine whether the screening protocol developed was appropriate. METHODS: From August 1998, we used liberalized screening criteria for patients who were prospectively identified and suspected to be at high risk for BCI/BVI if any of the following were present: anisocoria, unexplained mono-/hemiparesis, unexplained neurologic exam, basilar skull fracture through or near the carotid canal, fracture through the foramen transversarium, cerebrovascular accident or transient ischemic attack, massive epistaxis, severe flexion or extension cervical spine fracture, massive facial fractures, or neck hematoma. Four-vessel cerebral angiograms were used for screening for BCI/BVI. RESULTS: Over the 18-month study period, 48 patients were angiographically screened, with 21 patients (44%) being identified as having a total of 19 BCIs and 10 BVIs. Nine patients had unilateral carotid artery injuries and three patients had bilateral carotid artery injuries. Vertebral artery injuries were unilateral in six patients. One patient had bilateral carotid artery injuries and a unilateral vertebral artery injury. One patient had a unilateral carotid artery injury and a unilateral vertebral artery injury, and one patient had a unilateral carotid artery injury and bilateral vertebral artery injuries. During the same study period, 2,331 trauma patients were admitted, with 1,941 (83%) secondary to blunt trauma. The overall incidence of BCI/BVI was 1.1%. The frequency of abnormal angiograms ordered for cerebrovascular accident or transient ischemic attack, massive epistaxis, or severe cervical spine fractures was 100%. The frequency of abnormal angiograms ordered for the other indications was as follows: fracture through foramen transversarium, 60%; unexplained mono- or hemiparesis, 44%; basilar skull fracture, 42%; unexplained neurologic examination, 38%; anisocoria, 33%; and severe facial fractures, 0%. CONCLUSION: The liberalized screening criteria used in this study were appropriate to identify patients with BCI/BVI. This study suggests BCI/BVI to be more common than previously believed and justifies that screening should be liberalized.

Spontaneous renal artery dissection in a healthy man.

We have presented a unique case of isolated renal artery dissection in an otherwise healthy young man, whose diagnosis was demonstrated by renal angiography. He was anticoagulated with warfarin for one year with resolution of the false channel in his renal artery as demonstrated by magnetic resonance angiography. Duplex ultrasonography of his renal artery was important in monitoring his renal artery flow velocities.

Expression of the barley dehydrin multigene family and the development of freezing tolerance.

Dehydrins (DHNs; LEA D11) are one of the typical families of plant proteins that accumulate in response to dehydration, low temperature, osmotic stress or treatment with abscisic acid (ABA), or during seed maturation. We previously found that three genes encoding low-molecular-weight DHNs (Dhn1, Dhn2 and Dhn9) map within a 15-cM region of barley chromosome 5H that overlaps a QTL for winterhardiness, while other Dhn genes encoding low- and high-molecular-weight DHNs are located on chromosomes 3H, 4H and 6H. Here we examine the expression of specific Dhn genes under conditions associated with expression of the winterhardiness phenotype. Plants grown at 4 degrees C or in the field in Riverside, California developed similar, modest levels of freezing tolerance, coinciding with little low-MW Dhn gene activity. Dicktoo (the more tolerant cultivar) and Morex (the less tolerant) grown in Saskatoon, Canada expressed higher levels of expression of genes for low-MW DHNs than did the same cultivars in Riverside, with expression being higher in Dicktoo than Morex. Dehydration or freeze-thaw also evoked expression of genes for low MW DHNs, suggesting that the dehydration component of freeze-thaw in the field induces low expression of genes encoding low-MW DHNs. These observations are consistent with the hypothesis that the major chilling-induced DHNs help to prime plant cells for acclimation to more intense cold, which then involves adaptation to dehydration during freeze-thaw cycling. A role for chromosome 5H-encoded DHNs in acclimation to more intense cold seems possible, even though it is not the basis of the major heritable variation in winterhardiness within the Dicktoo x Morex population.