New Types of Wheat Chromosomal Structural Variations in Derivatives of Wheat-Rye Hybrids

Background

Chromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported.

Methodology/Principal Findings

Octoploid triticale lines were derived from common wheat Triticum. aestivum L. ‘Mianyang11’×rye Secale cereale L. ‘Kustro’. Some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ and common wheat T. aestivum L. ‘Chuannong27’ followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in ‘Mianyang11’ and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes.

Conclusions/Significance

These rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences.     

Molecular cytogenetic and genomic analyses reveal new insights into the origin of the wheat B genome

Key message

This work pinpointed the goatgrass chromosomal segment in the wheat B genome using modern cytogenetic and genomic technologies, and provided novel insights into the origin of the wheat B genome.

Abstract

Wheat is a typical allopolyploid with three homoeologous subgenomes (A, B, and D). The donors of the subgenomes A and D had been identified, but not for the subgenome B. The goatgrass Aegilops speltoides (genome SS) has been controversially considered a possible candidate for the donor of the wheat B genome. However, the relationship of the Ae. speltoides S genome with the wheat B genome remains largely obscure. The present study assessed the homology of the B and S genomes using an integrative cytogenetic and genomic approach, and revealed the contribution of Ae. speltoides to the origin of the wheat B genome. We discovered noticeable homology between wheat chromosome 1B and Ae. speltoides chromosome 1S, but not between other chromosomes in the B and S genomes. An Ae. speltoides-originated segment spanning a genomic region of approximately 10.46 Mb was detected on the long arm of wheat chromosome 1B (1BL). The Ae. speltoides-originated segment on 1BL was found to co-evolve with the rest of the B genome. Evidently, Ae. speltoides had been involved in the origin of the wheat B genome, but should not be considered an exclusive donor of this genome. The wheat B genome might have a polyphyletic origin with multiple ancestors involved, including Ae. speltoides. These novel findings will facilitate genome studies in wheat and other polyploids.

     

CAFTAN: a tool for fast mapping, and quality assessment of cDNAs

Background  

The German cDNA Consortium has been cloning full length cDNAs and continued with their exploitation in protein localization experiments and cellular assays. However, the efficient use of large cDNA resources requires the development of strategies that are capable of a speedy selection of truly useful cDNAs from biological and experimental noise. To this end we have developed a new high-throughput analysis tool, CAFTAN, which simplifies these efforts and thus fills the gap between large-scale cDNA collections and their systematic annotation and application in functional genomics.     

RNA-Seq quantification of the human small airway epithelium transcriptome

Background

The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated.

Results

We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes.

Conclusions

The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information.     

The Protein Disulfide Isomerase gene family in bread wheat (<Emphasis Type="Italic">T. aestivum L</Emphasis>.)

Background  

The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.     

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust

Key message

The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat.

Abstract

The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.     

Molecular characterization of vernalization loci VRN1 in wild and cultivated wheats

Background  

Variability of the VRN1 promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses (donor of B and D genomes of polyploid wheats) were investigated. Accessions of wild diploid (T. boeoticum, T. urartu) and tetraploid (T. araraticum, T. timopheevii) species were studied for the first time.     

Increased genomic prediction accuracy in wheat breeding using a large Australian panel

Key message

Genomic prediction accuracy within a large panel was found to be substantially higher than that previously observed in smaller populations, and also higher than QTL-based prediction.

Abstract

In recent years, genomic selection for wheat breeding has been widely studied, but this has typically been restricted to population sizes under 1000 individuals. To assess its efficacy in germplasm representative of commercial breeding programmes, we used a panel of 10,375 Australian wheat breeding lines to investigate the accuracy of genomic prediction for grain yield, physical grain quality and other physiological traits. To achieve this, the complete panel was phenotyped in a dedicated field trial and genotyped using a custom Axiom^TM Affymetrix SNP array. A high-quality consensus map was also constructed, allowing the linkage disequilibrium present in the germplasm to be investigated. Using the complete SNP array, genomic prediction accuracies were found to be substantially higher than those previously observed in smaller populations and also more accurate compared to prediction approaches using a finite number of selected quantitative trait loci. Multi-trait genetic correlations were also assessed at an additive and residual genetic level, identifying a negative genetic correlation between grain yield and protein as well as a positive genetic correlation between grain size and test weight.

     

A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block

Background  

The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features.     

Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker

Background  

Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.     

Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis

Background

Since the discovery of human immunodeficiency virus (HIV-1) twenty years ago, AIDS has become one of the most studied diseases. A number of viruses have subsequently been identified to contribute to the pathogenesis of HIV and its opportunistic infections and cancers. Therefore, a multi-virus array containing eight human viruses implicated in AIDS pathogenesis was developed and its efficacy in various applications was characterized.

Results

The amplified open reading frames (ORFs) of human immunodeficiency virus type 1, human T cell leukemia virus types 1 and 2, hepatitis C virus, Epstein-Barr virus, human herpesvirus 6A and 6B, and Kaposi's sarcoma-associated herpesvirus were spotted on glass slides and hybridized to DNA and RNA samples. Using a random priming method for labeling genomic DNA or cDNA probes, we show specific detection of genomic viral DNA from cells infected with the human herpesviruses, and effectively demonstrate the inhibitory effects of a cellular cyclin dependent kinase inhibitor on viral gene expression in HIV-1 and KSHV latently infected cells. In addition, we coupled chromatin immunoprecipitation with the virus chip (ChIP-chip) to study cellular protein and DNA binding.

Conclusions

An amplicon based virus chip representing eight human viruses was successfully used to identify each virus with little cross hybridization. Furthermore, the identity of both viruses was correctly determined in co-infected cells. The utility of the virus chip was demonstrated by a variety of expression studies. Additionally, this is the first demonstrated use of ChIP-chip analysis to show specific binding of proteins to viral DNA, which, importantly, did not require further amplification for detection.     

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4′-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-β−estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-β−estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.     

Exon definition as a potential negative force against intron losses in evolution

Background  

Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force.     

Characterisation of a gene encoding wheat endosperm starch branching enzyme-I

 A genomic DNA fragment from Triticum tauschii, the donor of the wheat D genome, contains a starch branching enzyme-I (SBE-I) gene spread over 6.5 kb. This gene (designated wSBE I-D4) encodes an amino acid sequence identical to that determined for the N-terminus of SBE-I from the hexaploid wheat (T. aestivum) endosperm. Cognate cDNA sequences for wSBE I-D4 were isolated from hexaploid wheat by hybridisation screening from an endosperm library and also by PCR. A contiguous sequence (D4 cDNA) was assembled from the sequence of five overlapping partial cDNAs which spanned wSBE I-D4. D4 cDNA encodes a mature polypeptide of 87 kDa that shows 90% identity to SBE-I amino acid sequences from rice and maize and contains all the residues considered essential for activity. D4 mRNA has been detected only in the endosperm and is at a maximum concentration mid-way through grain development. The wSBE I-D4 gene consists of 14 exons, similar to the structure for the equivalent gene in rice; the rice gene has a strikingly longer intron 2. The 3′ end of wSBE I-D4 was used to show that the gene is located on group 7 chromosomes. The sequence upstream of wSBE I-D4 was analysed with respect to conserved motifs. Received: 14 January 1998 / Accepted: 14 July 1998     

Genomic organization of ATOX1, a human copper chaperone

Background

Copper is an essential trace element that plays a critical role in the survival of all living organisms. Menkes disease and occipital horn syndrome (OHS) are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A) that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network. Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways. Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein. Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mo^br), a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported.

Results

We determined the genomic structure of ATOX1. The gene contains 4 exons spanning a genomic distance of approximately 16 kb. The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3. We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA. We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found.

Conclusions

In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport.

     

Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> 10403S

Background  

Information regarding the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively.