Comparative Genomics Tools Research Articles

A genetic linkage map of red drum, Sciaenops ocellatus

Second-generation, sex-specific genetic linkage maps were generated for the economically important estuarine-dependent marine fish Sciaenops ocellatus (red drum). The maps were based on F(1) progeny from each of two single-pair mating families. A total of 237 nuclear-encoded microsatellite markers were mapped to 25 linkage groups. The female map contained 226 markers, with a total length of 1270.9 centiMorgans (cM) and an average inter-marker interval of 6.53 cM; the male map contained 201 markers, with a total length of 1122.9 cM and an average inter-marker interval of 6.03 cM. The overall recombination rate was approximately equal in the two sexes (♀:♂=1.03:1). Recombination rates in a number of linkage intervals, however, differed significantly between the same sex in both families and between sexes within families. The former occurred in 2.4% of mapped intervals, while the latter occurred in 51.2% of mapped intervals. Sex-specific recombination rates varied within chromosomes, with regions of both female-biased and male-biased recombination. Original clones from which the microsatellite markers were generated were compared with genome sequence data for the spotted green puffer, Tetraodon nigroviridis; a total of 43 matches were located in 17 of 21 chromosomes of T. nigroviridis, while seven matches were in unknown portions of the T. nigroviridis genome. The map for red drum provides a new, useful tool for aquaculture, population genetics, and comparative genomics of this economically important marine species.

Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

BackgroundCryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains.ResultsThe majority of the putative species-specific genes examined were in fact common to C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes.ConclusionsMost of the genes identified as being species specific during bioinformatics in Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia.

Identification of genetic variation and haplotype structure of the canine ABCA4 gene for retinal disease association studies

Over 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype-phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed sequence diversity of ABCA4 in the dog, identifying 80% of novel variations. While none of the observed polymorphisms have been associated with blinding disorders to date, breed and potentially disease specific haplotypes have been identified. Moreover, a tag SNP map of 17 (15) markers has been established that accurately predicts common ABCA4 haplotypes (frequency > 5%) explaining >85% (>80%) of the observed genetic diversity and will considerably advance future studies. Our sequence analysis of the complete canine ABCA4 coding region will clearly provide a baseline and tools for future association studies and comparative genomics to further delineate the role of ABCA4 in canine blinding disorders.

Preventing preterm birth: the past limitations and new potential of animal models

The high rate of preterm birth in the USA and many other countries is a potential target for improving children's immediate health and reducing the medical problems they face as adults. The acute complications for infants born prematurely often require intensive care management and are followed by long-lasting cognitive, sensory, motor, and cardiovascular deficits that substantially limit adult capabilities and survival. The inability to effectively reduce preterm birth stems from the failure to understand normal mechanisms of parturition in humans. Although studies from several model organisms help define the physiology of maintenance and termination of pregnancy, there are fundamental differences between species. For example, species regulate their production of progesterone, the crucial hormone in sustaining pregnancy, differently. This limits the extent to which models can provide meaningful information about the physiological mechanisms of human gestation. The growing wealth of sequenced mammalian genomes, computational comparative genomic tools and systems biology approaches provides new potential to utilize the divergence of DNA sequences and physiology between species to understand the genetic underpinnings of preterm birth.

RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach

RegPredict web server is designed to provide comparative genomics tools for reconstruction and analysis of microbial regulons using comparative genomics approach. The server allows the user to rapidly generate reference sets of regulons and regulatory motif profiles in a group of prokaryotic genomes. The new concept of a cluster of co-regulated orthologous operons allows the user to distribute the analysis of large regulons and to perform the comparative analysis of multiple clusters independently. Two major workflows currently implemented in RegPredict are: (i) regulon reconstruction for a known regulatory motif and (ii) ab initio inference of a novel regulon using several scenarios for the generation of starting gene sets. RegPredict provides a comprehensive collection of manually curated positional weight matrices of regulatory motifs. It is based on genomic sequences, ortholog and operon predictions from the MicrobesOnline. An interactive web interface of RegPredict integrates and presents diverse genomic and functional information about the candidate regulon members from several web resources. RegPredict is freely accessible at http://regpredict.lbl.gov.

Pathline: A Tool For Comparative Functional Genomics

AbstractBiologists pioneering the new field of comparative functional genomics attempt to infer the mechanisms of gene regulation by looking for similarities and differences of gene activity over time across multiple species. They use three kinds of data: functional data such as gene activity measurements, pathway data that represent a series of reactions within a cellular process, and phylogenetic relationship data that describe the relatedness of species. No existing visualization tool can visually encode the biologically interesting relationships between multiple pathways, multiple genes, and multiple species. We tackle the challenge of visualizing all aspects of this comparative functional genomics dataset with a new interactive tool called Pathline. In addition to the overall characterization of the problem and design of Pathline, our contributions include two new visual encoding techniques. One is a new method for linearizing metabolic pathways that provides appropriate topological information and supports the comparison of quantitative data along the pathway. The second is the curvemap view, a depiction of time series data for comparison of gene activity and metabolite levels across multiple species. Pathline was developed in close collaboration with a team of genomic scientists. We validate our approach with case studies of the biologists’ use of Pathline and report on how they use the tool to confirm existing findings and to discover new scientific insights.

Cloud computing for comparative genomics

BackgroundLarge comparative genomics studies and tools are becoming increasingly more compute-expensive as the number of available genome sequences continues to rise. The capacity and cost of local computing infrastructures are likely to become prohibitive with the increase, especially as the breadth of questions continues to rise. Alternative computing architectures, in particular cloud computing environments, may help alleviate this increasing pressure and enable fast, large-scale, and cost-effective comparative genomics strategies going forward. To test this, we redesigned a typical comparative genomics algorithm, the reciprocal smallest distance algorithm (RSD), to run within Amazon's Elastic Computing Cloud (EC2). We then employed the RSD-cloud for ortholog calculations across a wide selection of fully sequenced genomes.ResultsWe ran more than 300,000 RSD-cloud processes within the EC2. These jobs were farmed simultaneously to 100 high capacity compute nodes using the Amazon Web Service Elastic Map Reduce and included a wide mix of large and small genomes. The total computation time took just under 70 hours and cost a total of $6,302 USD.ConclusionsThe effort to transform existing comparative genomics algorithms from local compute infrastructures is not trivial. However, the speed and flexibility of cloud computing environments provides a substantial boost with manageable cost. The procedure designed to transform the RSD algorithm into a cloud-ready application is readily adaptable to similar comparative genomics problems.

Rapid pair-wise synteny analysis of large bacterial genomes using web-based GeneOrder4.0

BackgroundThe growing whole genome sequence databases necessitate the development of user-friendly software tools to mine these data. Web-based tools are particularly useful to wet-bench biologists as they enable platform-independent analysis of sequence data, without having to perform complex programming tasks and software compiling.FindingsGeneOrder4.0 is a web-based "on-the-fly" synteny and gene order analysis tool for comparative bacterial genomics (ca. 8 Mb). It enables the visualization of synteny by plotting protein similarity scores between two genomes and it also provides visual annotation of "hypothetical" proteins from older archived genomes based on more recent annotations.ConclusionsThe web-based software tool GeneOrder4.0 is a user-friendly application that has been updated to allow the rapid analysis of synteny and gene order in large bacterial genomes. It is developed with the wet-bench researcher in mind.

A fast and automated solution for accurately resolving protein domain architectures

Accurate prediction of the domain content and arrangement in multi-domain proteins (which make up >65% of the large-scale protein databases) provides a valuable tool for function prediction, comparative genomics and studies of molecular evolution. However, scanning a multi-domain protein against a database of domain sequence profiles can often produce conflicting and overlapping matches. We have developed a novel method that employs heaviest weighted clique-finding (HCF), which we show significantly outperforms standard published approaches based on successively assigning the best non-overlapping match (Best Match Cascade, BMC). We created benchmark data set of structural domain assignments in the CATH database and a corresponding set of Hidden Markov Model-based domain predictions. Using these, we demonstrate that by considering all possible combinations of matches using the HCF approach, we achieve much higher prediction accuracy than the standard BMC method. We also show that it is essential to allow overlapping domain matches to a query in order to identify correct domain assignments. Furthermore, we introduce a straightforward and effective protocol for resolving any overlapping assignments, and producing a single set of non-overlapping predicted domains. The new approach will be used to determine MDAs for UniProt and Ensembl, and made available via the Gene3D website: http://gene3d.biochem.ucl.ac.uk/Gene3D/. The software has been implemented in C++ and compiled for Linux: source code and binaries can be found at: ftp://ftp.biochem.ucl.ac.uk/pub/gene3d_data/DomainFinder3/ yeats@biochem.ucl.ac.uk Supplementary data are available at Bioinformatics online.

Cost-Effective Cloud Computing: A Case Study Using the Comparative Genomics Tool, Roundup

BackgroundComparative genomics resources, such as ortholog detection tools and repositories are rapidly increasing in scale and complexity. Cloud computing is an emerging technological paradigm that enables researchers to dynamically build a dedicated virtual cluster and may represent a valuable alternative for large computational tools in bioinformatics. In the present manuscript, we optimize the computation of a large-scale comparative genomics resource—Roundup—using cloud computing, describe the proper operating principles required to achieve computational efficiency on the cloud, and detail important procedures for improving cost-effectiveness to ensure maximal computation at minimal costs.MethodsUtilizing the comparative genomics tool, Roundup, as a case study, we computed orthologs among 902 fully sequenced genomes on Amazon’s Elastic Compute Cloud. For managing the ortholog processes, we designed a strategy to deploy the web service, Elastic MapReduce, and maximize the use of the cloud while simultaneously minimizing costs. Specifically, we created a model to estimate cloud runtime based on the size and complexity of the genomes being compared that determines in advance the optimal order of the jobs to be submitted.ResultsWe computed orthologous relationships for 245,323 genome-to-genome comparisons on Amazon’s computing cloud, a computation that required just over 200 hours and cost $8,000 USD, at least 40% less than expected under a strategy in which genome comparisons were submitted to the cloud randomly with respect to runtime. Our cost savings projections were based on a model that not only demonstrates the optimal strategy for deploying RSD to the cloud, but also finds the optimal cluster size to minimize waste and maximize usage. Our cost-reduction model is readily adaptable for other comparative genomics tools and potentially of significant benefit to labs seeking to take advantage of the cloud as an alternative to local computing infrastructure.

HaloWeb: the haloarchaeal genomes database

BackgroundComplete genome sequencing together with post-genomic studies provide the opportunity for a comprehensive 'systems biology' understanding of model organisms. For maximum effectiveness, an integrated database containing genomic, transcriptomic, and proteomic data is necessary.DescriptionTo improve data access and facilitate functional genomic studies on haloarchaea in our laboratory, a dedicated database and website, named HaloWeb, was developed. It incorporates all finished and publicly released haloarchaeal genomes, including gene, protein and RNA sequences and annotation data, as well as other features such as insertion element sequences. The HaloWeb database was designed for easy data access and mining, and includes tools for tasks such as genome map generation, sequence extraction, and sequence editing. Popular resources at other sites, e.g., NCBI PubMed and BLAST, COG and KOG protein clusters, KEGG pathways, and GTOP structures were dynamically linked. The HaloWeb site is located at http://halo4.umbi.umd.edu, and at a mirror site, http://halo5.umbi.umd.edu, with all public genomic data and NCBI, KEGG, and GTOP links available for use by the academic community. The database is curated and updated on a regular basis.ConclusionsThe HaloWeb site includes all completely sequenced haloarchaeal genomes from public databases. It is currently being used as a tool for comparative genomics, including analysis of gene and genome structure, organization, and function. The database and website are up-to-date resources for researchers worldwide.

Stress-induced survival strategies enable Salmonella Enteritidis to persistently colonize the chicken oviduct tissue and cope with antimicrobial factors in egg white: A hypothesis to explain a pandemic

BackgroundEgg-associated transmission to humans seems to be characteristic of the Salmonella serotype Enteritidis, explaining why this particular serotype has caused a worldwide pandemic since the mid '80s. Salmonella Enteritidis is much more capable to persistently colonize the laying hen reproductive tract and to survive in the hostile egg white, as compared to other serotypes.Presentation of the hypothesisIt is hypothesized that stress-induced survival mechanisms enable the serotype Enteritidis to persistently colonize the oviduct without causing damage and excessive inflammation, and to cope with the antimicrobial compounds present in egg white.Testing the hypothesisTo test the hypothesis first of all Salmonella Enteritidis genes that are essential for colonization of the oviduct and survival in eggs need to be identified. Comparative genomics tools should be used to identify genes or pathogenicity islands that are present in Salmonella Enteritidis and not in the multiple non egg-contaminating serotypes. High-throughput signature-tagged-mutagenesis approaches, coupled to micro-array detection of the genes that lead to an attenuated phenotype when mutated is proposed as an ideal tool to identify genes involved in oviduct colonization and egg white survival. Identifying the stressors and antibacterial molecules in the oviduct and in the egg white that limit colonization or survival of non-Enteritidis serotypes is a second important objective that can theoretically be achieved using screenings of expressed oviduct cDNA libraries for their antibacterial activity against strains from multiple serotypes. Finally, the effect of contact with these stressors in the oviduct or egg white on Salmonella gene expression will need to be analyzed, in order to clarify whether serotype Enteritidis-specific regulation of certain stress-survival pathways are either or not present.Implications of the hypothesisKnowledge on the pathogenesis of egg infections would furthermore give insights that might be extrapolated to other biological interactions, in which a highly specialized bacterial pathogen resists the host response in a specific biological niche. In addition, this info can be of value in developing early warning criteria to identify emerging egg-associated Salmonella strains and in developing safe live attenuated vaccine strains.

Ureide catabolism in Arabidopsis thaliana and Escherichia coli

The availability of whole genome sequences boosts the identification of biochemical pathways conserved across species using tools of comparative genomics. A cross-organism protein association analysis allowed us to identify two enzymes, ureidoglycine aminohydrolase and ureidoglycolate amidohydrolase, that catalyze the final reactions of purine degradation in the model plant Arabidopsis thaliana. A similar pathway was found in Escherichia coli, while an alternative metabolic route via ureidoglycine transaminase can be predicted for other organisms.

Improved criteria and comparative genomics tool provide new insights into grass paleogenomics

In the past decade, a number of bioinformatics tools have been developed to perform comparative genomics studies in plants and animals. However, most of the publicly available and user friendly tools lack common standards for the identification of robust orthologous relationships between genomes leading non-specialists to often over interpret the results of large scale comparative sequence analyses. Recently, we have established a number of improved parameters and tools to define significant relationships between genomes as a basis to develop paleogenomics studies in grasses. Here, we describe our approaches and propose the development of community-based standards that can be used in comparative genomic studies to (i) identify robust sets of orthologous gene pairs, (ii) derive complete sets of chromosome to chromosome relationships within and between genomes and (iii) model common paleo-ancestor genome structures. The rice and sorghum genome sequences are used to exemplify step-by-step a methodology that should allow users to perform accurate comparative genome analyses in their favourite species. Finally, we describe two applications for accurate gene annotation and synteny-based cloning of agronomically important traits.

Comparative genomics allows the discovery of cis -regulatory elements in mosquitoes

The discovery and mapping of cis-regulatory elements is important for understanding regulation of gene transcription in mosquito vectors of human diseases. Genome sequence data are available for 3 species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus (Diptera: Culicidae), representing 2 subfamilies (Culicinae and Anophelinae) that are estimated to have diverged 145 to 200 million years ago. Comparative genomics tools were used to screen genomic DNA fragments located in the 5'-end flanking regions of orthologous genes. These analyses resulted in the identification of 137 sequences, designated "mosquito motifs," 7 to 9 nucleotides in length, representing 18 families of putative cis-regulatory elements conserved significantly among the 3 species when compared to the fruit fly, Drosophila melanogaster. Forty-one of the motifs were implicated previously in experiments as sites for binding transcription factors or functioning in the regulation of mosquito gene expression. Further analyses revealed associations between specific motifs and expression profiles, particularly in those genes that show increased or decreased mRNA abundance in females following a blood meal, and those accumulating transcription products exclusively or preferentially in the midgut, fat bodies, or ovaries. These results validate the methodology and support a relationship between the discovered motifs and the conservation of hematophagy in mosquitoes.

Location, Location, Location! Monotremes Provide Unique Insights into the Evolution of Sex Chromosome Silencing in Mammals

Platypus and echidnas are the only living representative of the egg-laying mammals that diverged 166 million years ago from the mammalian lineage. Despite occupying a key spot in mammalian phylogeny, research on monotremes has been limited by access to material and lack of molecular genetic resources. This has changed recently, and the sequencing of the platypus genome has promoted monotremes into a generally accessible tool in comparative genomics. The most extraordinary aspect of the monotreme genome is an amazingly complex sex chromosomes system that shares extensive homology with bird sex chromosomes and no homology with sex chromosomes of other mammals. This raises important questions about dosage compensation of the five pairs of sex chromosomes in females and meiotic silencing in males, and we are only beginning to unravel possible mechanisms and pathways that may be involved. The homology between monotreme and bird sex chromosomes makes comparison between those species worthwhile, also as they provide a well-defined example where the same sex chromosomes changed from female heterogamety (chicken) to male heterogamety (monotremes). We summarize recent research on monotreme and chicken sex chromosomes and discuss possible mechanisms that may contribute to sex chromosome silencing in monotremes.

The Moss Physcomitrella patens: A Novel Model System for Plant Development and Genomic Studies

INTRODUCTIONThe moss Physcomitrella patens has been used as an experimental organism for more than 80 years. Within the last 15 years, its use as a model to explore plant functions has increased enormously. The ability to use gene targeting and RNA interference methods to study gene function, the availability of many tools for comparative and functional genomics (including a sequenced and assembled genome, physical and genetic maps, and more than 250,000 expressed sequence tags [ESTs]), and a dominant haploid phase that allows direct forward genetic analysis have all led to a surge of new activity. P. patens can be easily cultured and spends the majority of its life cycle in the haploid state, allowing the application of experimental techniques similar to those used in microbes and yeast. Its development is relatively simple, and it generates only a few tissues that contain a limited number of cell types. Although mosses lack vascular tissue, true roots/stems/leaves, and flowers and seeds, many signaling pathways found in angiosperms are intact in moss. For example, the phytohormones auxin, cytokinin, and abscisic acid, as well as the photomorphogenic pigments phytochrome and cryptochrome, are all interwoven into distinct but overlapping pathways and linked to clear developmental phenotypes. In addition, about one-quarter of the moss genome contains genes with no known function based on sequence motifs, raising the likelihood of successful discovery efforts to identify new and novel gene functions.

A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): An important genetic resource for comparative genomics and phylogenetic analysis

Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.

MicroScope: a platform for microbial genome annotation and comparative genomics.

The initial outcome of genome sequencing is the creation of long text strings written in a four letter alphabet. The role of in silico sequence analysis is to assist biologists in the act of associating biological knowledge with these sequences, allowing investigators to make inferences and predictions that can be tested experimentally. A wide variety of software is available to the scientific community, and can be used to identify genomic objects, before predicting their biological functions. However, only a limited number of biologically interesting features can be revealed from an isolated sequence. Comparative genomics tools, on the other hand, by bringing together the information contained in numerous genomes simultaneously, allow annotators to make inferences based on the idea that evolution and natural selection are central to the definition of all biological processes. We have developed the MicroScope platform in order to offer a web-based framework for the systematic and efficient revision of microbial genome annotation and comparative analysis (http://www.genoscope.cns.fr/agc/microscope). Starting with the description of the flow chart of the annotation processes implemented in the MicroScope pipeline, and the development of traditional and novel microbial annotation and comparative analysis tools, this article emphasizes the essential role of expert annotation as a complement of automatic annotation. Several examples illustrate the use of implemented tools for the review and curation of annotations of both new and publicly available microbial genomes within MicroScope’s rich integrated genome framework. The platform is used as a viewer in order to browse updated annotation information of available microbial genomes (more than 440 organisms to date), and in the context of new annotation projects (117 bacterial genomes). The human expertise gathered in the MicroScope database (about 280,000 independent annotations) contributes to improve the quality of microbial genome annotation, especially for genomes initially analyzed by automatic procedures alone.Database URLs: http://www.genoscope.cns.fr/agc/mage and http://www.genoscope.cns.fr/agc/microcyc

Wet Lab Tools Widely Used in Plant Genomics

The availability of laboratory tools is essential for advanced research in all areas of biological sciences. The recent development of genomic tools has made it possible to deeply investigate and to continuously improve agronomically important traits such as crop yield, quality, and biotic and abiotic stress tolerances. Integrating the newly advanced or developed wet laboratory tools that are widely used in modern genomics research and making them readily accessible will be greatly helpful for research of plant genomics and other disciplines of plant biology. Furthermore, the compilation of the tools will also facilitate scientists to advance the existing tools or develop new tools to address complicated or new questions that were previously intractable in plant genomics and biology. In this special issue of the International Journal of Plant Genomics, “Wet lab tools widely used in plant genomics”, we present the current status of widely used genomics tools and update them with their new advances. Articles published in this special issue cover tools for structural, functional, and comparative genomics and proteomics. The issue also summarizes the advances of genome technology in the past decades and synthesizes the current status of knowledge of new tools with an extension of suggestions. By covering up-to-date genomics tools, this special issue provides a reference for studying the structural and functional organization and evolution of plant genomes. We aim that this special issue will become useful material for teaching and research in plant genomics and biology. There are 8 articles in this special issue, starting with articles on tools for studying plant authopagy (Mitou et al.) and microRNA identification (Unver and Budak), cloning of small RNA (Eric et al.), and the use of virus-induced gene silencing techniques (Unver et al.) for functional analysis of genes and QTLs in plants. Included is also a comprehensive article on heterologous gene expression techniques given by Filiz and Sayers. An up-to-date protocol of Agro-mediated gene transfer in cereal crops is presented by Hensel et al. Additionally, Dechyeva and Schmidt reported one of the critical tools, the molecular cytogenetic mapping of chromosomal fragments and immunostaining of kinetochore proteins, which will greatly help cytogeneticists for identifying and tagging genes in plants, thus promoting plant molecular breeding.