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The identification of mRNA isoforms in biological samples is crucial for studying tissue- and cell-specific isoform expression, activity of tissue-specific promoters, alternative splicing events, and alternative polyadenylation signals in genes. For single or several genes, expressed mRNA isoforms can be found using RT-PCR and RT-qPCR. Available transcriptome short-read archives deposited in GenBank or as laboratory data can be used to identify mRNA isoforms instead of or prior to wet analysis by other methods in eukaryotic organisms with annotated genomes. However, isoform expression analysis requires advanced bioinformatics skills and may be time-consuming. In addition, this analysis generates a large amount of unnecessary data. To detect mRNA isoforms encoded by one gene of interest, screening of expressed mRNAs in NGS data can be simplified by mapping NGS short reads to a single-gene or transcript sequence. Using single-gene/transcript mapping, we analyzed the expression of the Otx gene at the mRNA isoform level in some embryonic and adult tissue mRNA libraries of the sea urchin Strongylocentrotus purpuratus available in GenBank. The presence of expressed Otx mRNA isoforms was confirmed by RT-qPCR in the same tissues and at the same developmental stages of the closely related species Strongylocentrotus intermedius. We showed that single-gene/transcript mapping is a suitable approach for qualitative evaluation of the expression of mRNA isoforms and recognition of at least two expressed isoforms in the same biological sample.

Of central importance in adapting plants of tropical origin to temperate cultivation has been selection of daylength-neutral genotypes that flower early in the temperate summer and take full advantage of its long days. A cross between tropical and temperate sorghums [Sorghum propinquum (Kunth) Hitchc.×S. bicolor (L.) Moench], revealed a quantitative trait locus (QTL), FlrAvgD1, accounting for 85.7% of variation in flowering time under long days. Fine-scale genetic mapping placed FlrAvgD1 on chromosome 6 within the physically largest centiMorgan in the genome. Forward genetic data from “converted” sorghums validated the QTL. Association genetic evidence from a diversity panel delineated the QTL to a 10-kb interval containing only one annotated gene, Sb06g012260, that was shown by reverse genetics to complement a recessive allele. Sb06g012260 (SbFT12) contains a phosphatidylethanolamine-binding (PEBP) protein domain characteristic of members of the “FT” family of flowering genes acting as a floral suppressor. Sb06g012260 appears to have evolved ∼40 Ma in a panicoid ancestor after divergence from oryzoid and pooid lineages. A species-specific Sb06g012260 mutation may have contributed to spread to temperate regions by S. halepense (“Johnsongrass”), one of the world’s most widespread invasives. Alternative alleles for another family member, Sb02g029725 (SbFT6), mapping near another flowering QTL, also showed highly significant association with photoperiod response index (P = 1.53×10 − 6). The evolution of Sb06g012260 adds to evidence that single gene duplicates play large roles in important environmental adaptations. Increased knowledge of Sb06g012260 opens new doors to improvement of sorghum and other grain and cellulosic biomass crops.

Postemergence grass weed control continues to be a major challenge in grain sorghum [ Sorghum bicolor (L.) Moench], primarily due to lack of herbicide options registered for use in this crop. The development of herbicide-resistant sorghum technology to facilitate broad-spectrum postemergence weed control can be an economical and viable solution. The 4-hydroxyphenylpyruvate dioxygenase-inhibitor herbicides (e.g., mesotrione or tembotrione) can control a broad spectrum of weeds including grasses, which, however, are not registered for postemergence application in sorghum due to crop injury. In this study, we identified two tembotrione-resistant sorghum genotypes (G-200, G-350) and one susceptible genotype (S-1) by screening 317 sorghum lines from a sorghum association panel (SAP). These tembotrione-resistant and tembotrione-susceptible genotypes were evaluated in a tembotrione dose–response [0, 5.75, 11.5, 23, 46, 92 (label recommended dose), 184, 368, and 736 g ai ha –1 ] assay. Compared with S-1, the genotypes G-200 and G-350 exhibited 10- and seven fold more resistance to tembotrione, respectively. To understand the inheritance of tembotrione-resistant trait, crosses were performed using S-1 and G-200 or G-350 to generate F 1 and F 2 progeny. The F 1 and F 2 progeny were assessed for their response to tembotrione treatment. Genetic analyses of the F 1 and F 2 progeny demonstrated that the tembotrione resistance in G-200 and G-350 is a partially dominant polygenic trait. Furthermore, cytochrome P450 (CYP)-inhibitor assay using malathion and piperonyl butoxide suggested possible CYP-mediated metabolism of tembotrione in G-200 and G-350. Genotype-by-sequencing based quantitative trait loci (QTL) mapping revealed QTLs associated with tembotrione resistance in G-200 and G-350 genotypes. Overall, the genotypes G-200 and G-350 confer a high level of metabolic resistance to tembotrione and controlled by a polygenic trait. There is an enormous potential to introgress the tembotrione resistance into breeding lines to develop agronomically desirable sorghum hybrids.

Background Chromosomal variations have been revealed in both E. sibiricus and E. nutans , but chromosomal structural variations, such as intra-genome translocations and inversions, are still not recognized due to the cytological limitations of previous studies. Furthermore, the syntenic relationship between both species and wheat chromosomes remains unknown. Results Fifty-nine single-gene fluorescence in situ hybridization (FISH) probes, including 22 single-gene probes previously mapped on wheat chromosomes and other newly developed probes from the cDNA of Elymus species, were used to characterize the chromosome homoeologous relationship and collinearity of both E. sibiricus and E. nutans with those of wheat. Eight species-specific chromosomal rearrangements (CRs) were exclusively identified in E. sibiricus , including five pericentric inversions in 1H, 2H, 3H, 6H and 2St; one possible pericentric inversion in 5St; one paracentric inversion in 4St; and one reciprocal 4H/6H translocation. Five species-specific CRs were identified in E. nutans , including one possible pericentric inversion in 2Y, three possible pericentric multiple-inversions in 1H, 2H and 4Y, and one reciprocal 4Y/5Y translocation. Polymorphic CRs were detected in three of the six materials in E. sibiricus , which were mainly represented by inter-genomic translocations. More polymorphic CRs were identified in E. nutans , including duplication and insertion, deletion, pericentric inversion, paracentric inversion, and intra- or inter-genomic translocation in different chromosomes. Conclusions The study first identified the cross-species homoeology and the syntenic relationship between E. sibiricus , E. nutans and wheat chromosomes. There are distinct different species-specific CRs between E. sibiricus and E. nutans , which may be due to their different polyploidy processes. The frequencies of intra-species polymorphic CRs in E. nutans were higher than that in E. sibiricus . To conclude, the results provide new insights into genome structure and evolution and will facilitate the utilization of germplasm diversity in both E. sibiricus and E. nutans .

Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat ( Triticum aestivum ), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops . The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata , respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii . To develop molecular markers with exact physical positions on chromosomes of Aegilops , the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata . Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat- Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.

KEY MESSAGE : A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed. Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F₂ population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.

Using genome-wide information to understand holistically how cells function is a major challenge of the postgenomic era. Recent efforts to understand molecular pathway operation from a global perspective have lacked experimental data on phenotypic context, so insights concerning biologically relevant network characteristics of key genes or proteins have remained largely speculative. Here, we present a global network investigation of the genotype/phenotype data set we developed for the recovery of the yeast Saccharomyces cerevisiae from exposure to DNA-damaging agents, enabling explicit study of how protein-protein interaction network characteristics may be associated with phenotypic functional effects. We show that toxicity-modulating proteins have similar topological properties as essential proteins, suggesting that cells initiate highly coordinated responses to damage similar to those needed for vital cellular functions. We also identify toxico-logically important protein complexes, pathways, and modules. These results have potential implications for understanding toxicity-modulating processes relevant to a number of human diseases, including cancer and aging.

Background Plant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape ( Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2 . Results The inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2 , 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F 2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2 , which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture. Conclusions A dominant dwarfing gene, BnaC04.BIL1 , encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.

The fresh water polyp Hydra L., 1758 (Cnidaria, Hydrozoa) plays a key role as a model organism in modern evolutionary and developmental biology. A complete genome sequence has been published recently for Hydra magnipapillata Ito, 1947 and molecular data are rapidly accumulating in the literature, but little information is available on its chromosomes. In this study, an efficient fluorescence in situ hybridization (FISH) method is described for H. magnipapillata which not only allows identification of the chromosomes but also visualization of the location of individual genetic loci. Together with cDNA and genomic sequencing this may provide the foundation for increasingly precise genetic and physical mapping in this basal metazoan model organism.

A genetic model is developed with additive and dominance effects of a single gene and polygenes as well as general and specific reciprocal effects for the progeny from a diallel mating design. The methods of ANOVA, minimum norm quadratic unbiased estimation (MINQUE), restricted maximum likelihood estimation (REML), and maximum likelihood estimation (ML) are suggested for estimating variance components, and the methods of generalized least squares (GLS) and ordinary least squares (OLS) for fixed effects, while best linear unbiased prediction, linear unbiased prediction (LUP), and adjusted unbiased prediction are suggested for analyzing random effects. Monte Carlo simulations were conducted to evaluate the unbiasedness and efficiency of statistical methods involving two diallel designs with commonly used sample sizes, 6 and 8 parents, with no and missing crosses, respectively. Simulation results show that GLS and OLS are almost equally efficient for estimation of fixed effects, while MINQUE (1) and REML are better estimators of the variance components and LUP is most practical method for prediction of random effects. Data from a Drosophila melanogaster experiment (Gilbert 1985a, Theor appl Genet 69:625-629) were used as a working example to demonstrate the statistical analysis. The new methodology is also applicable to screening candidate gene(s) and to other mating designs with multiple parents, such as nested (NC Design I) and factorial (NC Design II) designs. Moreover, this methodology can serve as a guide to develop new methods for detecting indiscernible major genes and mapping quantitative trait loci based on mixture distribution theory. The computer program for the methods suggested in this article is freely available from the authors.