Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants.

If I had not been so lazy, I never would have realized that what turned out to be the most important experiment in my scientific career had actually went well. Indeed, for the first time in plants, I had just enzymatically induced a unique site-specific genomic double-strand break (DSB). From all we knew, this should have been the key to the controlled induction of various types of genomic rearrangements. However, there was no indication that the experiment had worked. But let us start from the beginning. During my study of biochemistry in the early eighties of the last millennium at the University of Tübingen in Germany, I became more and more interested in molecular biology and especially in gene technology. Although already in worldwide use for some time, it was hard to find groups in Germany that were using methods of gene technology routinely in the laboratory. For me, the opportunity to get involved came at the Max Plank Institute in Martinsried near Munich, where I earned my diploma and wrote my PhD thesis in the department of the late Heinz-Ludwig Sänger, one of the co-discoverers of viroids. Viroids are small, circular, single-stranded RNA molecules measuring 250–400 nucleotides in length that are able to infect plants and cause somewhat severe disease symptoms. During my time in laboratory, we cloned cDNA out of viroids, which turned out to be infectious when inoculated on plants. This was an amazing demonstration of the power of gene technology. During my PhD, I also discovered a new viroid that we found distributed in hops all over the globe without causing symptoms. Indeed, this was the only time in my career that my research was broadly covered by daily newspapers, but alas this was mainly because some journalist who craved sensationalism speculated that the worldwide distribution of an infectious agent in the hop plant might be a serious health threat for millions of beer consumers, especially in Bavaria. The small size of viroids made them for me no promising objects for gene technology in a long-term perspective. Therefore, my interest in the transformation of plants grew. At that time, the Friedrich Miescher Institute (FMI) in Basel, Switzerland, was one of the leading centres of plant molecular biology in Europe. There, Barbara Hohn, who was already well known as a pioneer of molecular biology due to her invention of the cosmids, was analysing the mechanism of Agrobacterium-mediated transformation in plants. Although it seems crazy to me today, after I finished my PhD, I sent out only a single job application, and it was to join her laboratory. Things worked out well: I got the position and stayed in her laboratory for the next five and half years, which turned out to be the most interesting, productive and formative time of my scientific career. At that time, Barbara became interested in studying plant genome stability. A main focus of my work was to set up a scorable assay system for measuring the frequency of homologous recombination (HR) between repetitive genomic sequences. The assay was initially developed for Arabidopsis (Swoboda et al., 1994) and tobacco and was based on the restoration of the ß-glucuronidase gene from overlapping nonfunctional parts. Over the years, the assay became a very valuable tool for plant biologists. It could not only be used to characterize the roles of individual proteins in genome stability in plants but also served as an assay that could define stress factors that challenge genome stability. Fortuitously, the setting up of this assay system also enabled us to prove that T-DNA is indeed transferred from Agrobacterium into plant cells as a single-stranded molecule (Tinland et al., 1994). During my stay in Basel, I became more and more interested in DNA recombination mechanisms (Figure 1). For all we knew, recombination reactions were initiated by DSBs in the DNA. Unfortunately, there was no way to induce a unique DSB in vivo at a specific position in the genome of a multicellular eukaryotic organism at that time. In studying the literature, I learned that Bernard Dujon's group at the Institute Pasteur had discovered a special type of sequence-specific endonuclease: the homing endonuclease I-SceI, which has an 18-mer recognition site. This site was long enough to be unique if transformed into smaller plant genomes. Statistically, the same sequence is not expected to occur naturally within such genomes. I-SceI exists in yeast mitochondria; its ORF is contained within a ribosomal RNA gene of mitochondrial DNA. The induction of a DSB can subsequently induce HR, allowing the endonuclease gene to spread into mitochondrial genomes that do not yet harbour its ORF (Jacquier and Dujon, 1985). I obtained a codon-optimized ORF from Bernard and performed pilot experiments with plasmid DNAs transfected into plant protoplasts. The result indicated that I-SceI can be used in plant cells to induce DSBs into plasmid DNAs. HR between different plasmid molecules could be enhanced by in vivo induction of such DSBs drastically (Puchta et al., 1993). Indeed, ours was the first publication demonstrating this approach in any multicellular eukaryote. However, the question remained: could a mitochondrial enzyme cut nuclear DNA that is complexed with chromatin? Furthermore, would the induction of DSBs indeed enhance the integration of a specific piece of transgenic DNA within a locus that carries the same sequence information ['gene targeting' (GT)]? At that time, GT was the Holy Grail of gene technology: a way to knock out gene function or integrate DNA into a specific genomic position. With the exception of mice, GT was not yet an established technique in most multicellular eukaryotes, including plants. Years earlier, at the ETH Zürich, Jurek Paszkowski had demonstrated that GT was achievable in plants, but only at a very low frequency (Paszkowski et al. 1988). I set up the respective experiments by transforming parts of an artificial target locus including an I-SceI site into tobacco (Figure 2a). As a marker that should be restored by HR, Jurek provided me with an artificial kanamycin resistance gene that contained a eukaryotic intron sequence to extend the length of homology in the targeting experiment. I obtained plants that contained single copies of the target locus and then retransformed them with two Agrobacterium strains that contained one T-DNA each, one with an expression cassette of I-SceI and the other with the targeting vector that included a part of the kanamycin resistance gene that was homologous to the target locus. The basic idea was that if we could indeed induce a DSB at the target locus via I-SceI expression, then multiple kanamycin-resistant calli should arise. However, to my great disappointment, at the time point during which we would normally observe resistant calli following transformation, I did not see anything growing on my plates. A week later and nothing again. Under normal circumstances, enough time had elapsed to warrant taking the plates out of the incubator and throwing them out; however, as I was lazy, I instead just left them there. After shifting them to the very back of the growth chamber, I forgot about them. Barbara had a busy laboratory with little room to spare. Thus, 2 weeks later, my colleagues came to tell me that space was needed and that I should remove my old plates. I took them out, and there they were as follows: green calli on all of the plates! The experiment had worked. We were able to enhance targeting via DSB induction by at least two orders of magnitude! We subjected the recombinants to molecular analysis, and most of them turned out to result from HR of the target vector with both ends of the genomic DSB. The change proved to be heritable and segregated in a Mendelian manner (Puchta et al., 1996). So, why was I unable to obtain resistant calli earlier? The intron-containing kanamycin gene was not as efficient at conferring resistance as the intronless gene that we normally used, which increased the length of time that was required to grow the resistant calli. Thus, if I had thrown the plates away 'in time', I would have thrown away my future. The time came to leave Barbara's group. Of the upmost importance to my future career was the fact that Barbara generously agreed that I could take all of my work related to DSB induction with me. I began looking for grant money to set up a junior research group. Of course, included in this search was the question of where to go. I applied for funding in both Switzerland and Germany and was fortunate enough to obtain grants that would allow me to work in either country. It is notable that my salary in Switzerland would have been about twice as high as in Germany. Nevertheless, I decided to leave the country, because with Barbara's and Jurek's groups, two worldwide leaders in the field of DNA recombination were already situated in Switzerland, whereas no group with such an expertise existed in Germany. I decided to give up the money and go for the better perspective by returning to Germany. Actually, I never 'returned' in the true sense of the word but instead came to a country that I had never been in before. When I had originally left West Germany, there were two independent states and the Berlin wall was still standing. When I came back, there was one unified Germany, and I ended up in the eastern part. This was a possibility that would not have entered my wildest dreams before I left. The institute in Gatersleben was 'the' premium place for plant genetics in the former GDR. After the unification, it became the Leibnitz Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK). I joined the department of cytogenetics, which was headed by Ingo Schubert, one of the leading European scientists in the field. At Gatersleben, I was in a very comfortable position as a young group leader. I had the unique tool of in vivo DSB induction in hand. At that time, nobody else in the world was using such an approach in plants. My tool could not only elucidate mechanisms of DSB repair but could also facilitate the development of new techniques for genome engineering. Indeed, our very first study turned out to already be very rewarding. We analysed DSB repair via nonhomologous end joining (NHEJ) using transgenic tobacco that contained a negative selectable marker gene with an I-SceI site and showed that the induction of DSBs enabled the ORF to be destroyed by NHEJ repair (Figure 2b). We found that some instances of NHEJ led to microhomologies at the newly formed junctions, whereas others did not, indicating that there are two different mechanisms of NHEJ. In addition to deletions, we also found a number of alleles bearing insertions. We found sequences copied from elsewhere in the genome into the DSB, as well as the integration of T-DNAs into the DSB (Figure 2c). Thus, we were able to show that DSB repair is a mechanism of T-DNA integration (Salomon and Puchta, 1998). Later, we performed similar experiments in Arabidopsis and found that species-specific differences exist during NHEJ repair. We suggested that the deletions associated with NHEJ might be a mechanism for the shrinking of plant genomes (Kirik et al., 2000), a hypothesis which has since been supported by bioinformatics analysis. We started a series of experiments that showed that DSB-induced HR is most efficient with sequences that are close to each other on the same chromosome and very inefficient if the sequences are in an allelic or ectopic position. Furthermore, we also developed tools for DSB-induced genome engineering. Thus, we were able to show that by inducing two DSBs in close proximity to each other, the intervening sequences can be removed from the genome (Figure 2d, Siebert and Puchta, 2002). We were later able to demonstrate that the reciprocal exchange of chromosomal arms can be achieved by inducing DSBs in the respective chromosomes. In addition to studying DSB repair itself, I developed a second target for our group, which was to characterize the proteins involved in DSB repair and genome stability in Arabidopsis. This proved to be a rewarding decision, as only a few groups in the world were working on what was quite a wide topic. As my space in this article is limited, I will focus on only the most important discoveries. We found a unique type of topoisomerase in plants, Top6, which descended from archaea and is involved in endoreduplication (Hartung et al., 2002). We also discovered a new type of mechanism for how meiotic recombination intermediates are dissolved by topoisomerase 3-alpha (Hartung et al., 2008), which might be general to all eukaryotes. Indeed, over the years, this work became the major focus of our group, particularly after I left Gatersleben to become chair of the department of plant molecular biology and biochemistry at the University of Karlsruhe, which is one of the leading technical universities of Germany and after merging with a federal research institute later became the Karlsruhe Institute of Technology (KIT). During this time, a new development began that transformed molecular biology: the construction of artificial nucleases. It all started with the development of zinc finger nucleases (ZNFs). It could be shown that by manipulation of the zinc finger containing DNA binding motives of transcription factors, new binding specificities could be obtained that did not exist in nature before (Rebar and Pabo, 1994). By fusing the nuclease domain of a restriction enzyme to such a DNA-binding domain artificial, programmable nucleases could be constructed (Kim et al., 1996; Smith et al., 2000). Later on, transcription activator-like effector nucleases (TALENS) were developed according to the same principle relaying on a different kind of DNA binding motive originating from a plant pathogenic bacterium (Boch et al., 2009). These synthetic enzymes were applied for the induction of genomic DSBs throughout the plant genome. Thus, the knockout (Lloyd et al., 2005) or GT (Shukla et al., 2009; Townsend et al., 2009; Wright et al., 2005) of any natural gene became a possibility for plants. The biggest leap forward, however, happened just 3 years ago when the CRISPR/Cas system was discovered as an efficient tool for genome engineering (Jinek et al., 2012). This technology made it possible to create a nuclease in an extremely easy way by simply cloning new guide RNAs to define the specificity of the Cas9 nuclease. Using multiple guide RNAs, we are now also able to simultaneously induce a larger number of DSBs within the plant genome. To me, the field of plant genome engineering has never been more exciting than it is now. At the current moment, thousands of scientists induce DSBs using artificial nucleases in various plant species to produce mutants that they would hardly be able to obtain by any other means. We recently developed a new type of GT strategy, 'in planta gene targeting', that makes large-scale transformation and tissue culture efforts obsolete (Fauser et al., 2012) because the GT reaction can take place not only shortly after transformation but also throughout the complete life cycle of a plant. Using CRISPR/Cas, we demonstrated that in planta, GT could be applied to natural genes in Arabidopsis (Schiml et al., 2014). We were also able to show that we can efficiently induce single-strand breaks (SSBs) in the plant genome using a modified Cas9 nuclease. For the first time, we are now able to analyse in detail the different mechanisms behind SSB repair in plants, and we can use the paired induction of two SSBs for mutant generation (to avoid the off-target effects of the classical CRISPR/Cas system). Because detailed knowledge of the factors involved in DSB repair is now available, we will be able to develop more sophisticated genome engineering techniques by combining DSB induction with specific DNA repair phenotypes. At the moment, we are just now becoming able to restructure genomes within a species; however, in the long run, we may indeed be able to create synthetic plant genomes. First and foremost, I must thank Barbara Hohn; without her continuous support, I would not have been able to develop a scientific career studying DSB repair. I also thank Ingo Schubert, who enthusiastically supported my research at the IPK. I am extraordinarily indebted to all of my motivated co-workers, without whom not a single experiment would have come to fruition, and to my friends in the plant DNA repair/recombination community for all of our rewarding interactions. With gratitude, I also acknowledge the funding agencies that supported my work, especially the DFG, the BMBF, the EU and the ERC. Last but by no means least, I would like to thank my wife, Waltraud Schmidt-Puchta, who is a scientist herself, for giving me the opportunity to develop my career while taking care of the most important project of our lives: our kids.

The field of telomere biology was pioneered, in large part, through the seminal studies of Barbara McClintock in maize more than 60 years ago. Although many of the key insights in telomere research in the ensuing years have derived from other organisms, there is renewed interest in plant telomere biology as our understanding of the intimate relationship between cellular proliferation and telomere maintenance evolves. This interest is fueled by the striking contrast between the plasticity of plant development and genome architecture, and the much more deterministic nature of mammals. In contrast to mammals, plants produce new organs throughout their lives from meristematic proliferation zones, and many cells in the plant body are totipotent. The plant genome also displays an exceptional plasticity and tolerance to genome stresses, including changes in ploidy, DNA methylation, chromosomal rearrangements, and transposition. Taken together, these features raise fundamental questions about the contribution of telomeres and telomerase in facilitating cell proliferation and genome stability in plants. In the last few years, plant telomere biology has begun to blossom, owing in large part to research with the small flowering plant of the mustard weed family, Arabidopsis thaliana . The completion of the Arabidopsis genome sequencing project and the availability of T-DNA insertion lines that allow for gene knockouts have enabled researchers to make rapid progress in deciphering the functions of several telomere-related genes and in uncovering plant responses to telomere dysfunction. In this review, I focus on recent studies of telomere architecture and function and illustrate how plants are...

The wondrous cycles of polyploidy in plants.

Human centromeres are genomic regions that act as sites of kinetochore assembly to ensure proper chromosome segregation during mitosis and meiosis. Although the biological importance of centromeres in genome stability, and ultimately, cell viability are well understood, the complete sequence content and organization in these multi-megabase-sized regions remains unknown. The lack of a high-resolution reference assembly inhibits standard bioinformatics protocols, and as a result, sequence-based studies involving human centromeres lag far behind the advances made for the non-repetitive sequences in the human genome. In this chapter, I introduce what is known about the genomic organization in the highly repetitive regions spanning human centromeres, and discuss the challenges these sequences pose for assembly, alignment, and data interpretation. Overcoming these obstacles is expected to issue a new era for centromere genomics, which will offer new discoveries in basic cell biology and human biomedical research.

The centromere is the chromosomal region where the kinetochore forms. The kinetochore is a protein structure mediating contacts with the mitotic and meiotic spindle microtubule structures, enabling separation of chromosomes in mitosis and meiosis. Failure of the chromosome segregation machinery either by inactivation of the centromere or malfunction of the kinetochore or spindle results in chromosome mis-segregation and aneuploidy of the daughter cells. Aneuploidy is associated with birth defects or cancer. Genome stability can also be compromised by activation of extra centromeres (neocentromeres). Thus, to ensure euploidy and correct chromosome segregation, epigenetic activation of a single centromere per chromosome is required. The centromere is marked epigenetically by incorporation of the histone H3 variant CENP-A in the centromeric chromatin. In most organisms endogenous centromeres are surrounded and stabilized by adjacent heterochromatin, together constituting regional centromeres. In some organisms, such as budding yeast, point centromeres of a single nucleosome exist without pericentric heterochromatin. Yet, in other organisms, for example the worm C. elegans, the entire chromosome forms a holocentric centromere [1]. The centromere and kinetochore proteins are highly conserved in eukaryotes and the underlying DNA sequence is composed of complex repeats in most organisms. By contrast with the conserved proteins, the repetitive sequences are highly variable, both between species and individuals [2]. For technical reasons, endogenous human centromeres have been challenging to study and most of the mechanistic insights come from studies of human artificial chromosomes or centromeres in genetically amenable model organisms, such as budding and fission yeasts and the fruit fly. In fission yeast for example, the borders between CENP-A chromatin and the surrounding heterochromatin are clearly defined and the underlying DNA repeats are distinct. However, in human cells, the alpha satellite repeats make up the majority of the centromere DNA and heterochromatin is interspersed with CENP-A chromatin. Recent technical advances such as the delineation of the human centromere repeats, superresolution microscopy and the proximity ligation assay allow us to embark on more detailed examinations of the regulation of the human centromere and how it might contribute to disease. For an acentric chromosome fragment to be propagated through somatic cell divisions and through the germ line of a eukaryotic organism, a new centromere needs to form. A so-called ‘neocentromere’ is a functional centromere structure that forms at a nontypical genomic region and at DNA sequences that may differ from endogenous centromeres [3]. Such neocentromeres have been observed to spontaneously occur in several cancers characterized by genetically instability [4] and, occasionally, in cells that have gone through many cell divisions. Alternatively, the centromere formation can be studied experimentally by artificially removEpigenetic activation and inactivation of centromeres

The plant genome: an evolutionary perspective on structure and function

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171 bp monomers at normal centromeres, and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA wrapping behavior as octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on their conventional counterparts containing canonical H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

Spermatogenesis is a tightly coordinated differentiation program that sustains male fertility while transmitting genetic and epigenetic information to the next generation. This review consolidates mechanistic evidence showing how RNA-centered regulation integrates with the epitranscriptome and three-dimensional (3D) genome architecture to orchestrate germ-cell fate transitions from spermatogonial stem cells through meiosis and spermiogenesis. Recent literature is critically surveyed and synthesized, with particular emphasis on human and primate data and on stage-resolved maps generated by single-cell and multi-omics technologies. Collectively, available studies support a layered regulatory model in which RNA-binding proteins and RNA modifications coordinate transcript processing, storage, translation, and decay; small and long noncoding RNAs shape post-transcriptional programs and transposon defense; and dynamic chromatin remodeling and 3D reconfiguration align transcriptional competence with recombination, sex-chromosome silencing, and genome packaging. Convergent nodes implicated in spermatogenic failure are highlighted, including defects in RNA metabolism, piRNA pathway integrity, epigenetic reprogramming, and nuclear architecture, and the potential of these frameworks to refine molecular phenotyping in male infertility is discussed. Finally, key gaps and priorities for causal testing in spatially informed, stage-specific experimental systems are outlined.

Genomic and functional variation of human centromeres

Duplicated eukaryotic chromosomes are segregated into daughter cells through cell division. Faithful chromosome segregation depends on kinetochores, which are specialized macromolecular structures built upon centromeric chromatin. The dynamic kinetochore structures connect chromosomes with spindle microtubules, power chromosome movement, and signal the activation and silencing of the spindle assembly checkpoint (SAC). Molecular analyses of the components and architecture of kinetochores have advanced rapidly in recent years. A human kinetochore contains approximately 200 proteins, many of which are evolutionarily conserved in other organisms. A histone H3 variant, CENP‐A and associated constitutive centromere proteins lay the foundation for kinetochore build‐up. Multiple kinetochore‐localised microtubule‐binding proteins including the Ndc80 complex help regulate chromosome movement. The SAC signalling originates from kinetochores and contributes to the fidelity of chromosome segregation. Many fascinating properties remain to be elucidated about the kinetochore as a fundamental machinery to maintain genomic stability. Key Concepts: Chromosome segregation in eukaryotic cells depends upon connecting spindle microtubules with special macromolecular structures on chromosomes called kinetochores. The centromere is the chromosomal locus where a kinetochore is built. Laying the foundation for kinetochore assembly at centromeres are CENP‐A (a histone H3 variant) containing nucleosomes and a group of CENP‐A associated proteins (termed constitutive centromere proteins). There are multiple microtubule motors and nonmotor microtubule‐binding proteins localised at kinetochores to coordinate chromosome movement. A 10 protein complex called KMN network is currently thought to provide the primary end‐on microtubule‐binding activity. The spindle assembly checkpoint (SAC) monitors the kinetochore–microtubule attachment and signals the delay of the metaphase‐to‐anaphase transition when defects are detected. Conformational change of MAD2 and assembly of the mitotic checkpoint complex (MCC) are the key events to activate the SAC. Comparative studies of similar and distinct kinetochore composition, structure and function in different species and during mitosis or meiosis have provided evolutionary perspectives on mechanisms regulating chromosome segregation.

This review asks how variation in genome architecture impacts speciation across the plant and animal kingdoms. First, we briefly summarize what is known about speciation in these groups; importantly, the diversification rate of plants is about twice that of animals, and species barriers in plants appear to arise at an earlier stage of divergence. Next, we discuss several of the major differences in how plant and animal genomes evolve, and how they may impact the evolution of reproductive barriers and potentially speciation rates. Key differences include (1) the higher frequency of whole genome duplications and more rapid loss of synteny in plants; (2) the higher incidence and greater divergence of sex chromosomes in animals; (3) higher rates of sequence change, but slower rates of structural evolution, in animal relative to plant mitochondrial genomes; and (4) the higher abundance of transposable elements in plant genomes. Overall, we find the genomes of plants typically diverge much more rapidly in structure than those of animals (although there are many exceptions), which likely contributes to the more rapid emergence reproductive barriers in plants. However, we also found that comparisons of genome evolution between the kingdoms are hampered by inconsistency in the methods employed, and in the metrics used to report on rates of structural evolution. Another theme from our review is the huge variation in genome architecture within each kingdom. While this variation complicates broad generalizations, it also enables powerful comparative analyses that link differences in genome architecture to patterns and processes of speciation.

ABSTRACTThere have been numerous treatments of specific topics in speciation, but surprisingly few papers have compared patterns and processes of speciation across different organismal groups. In this review, we partially address this gap by asking how variation in genome architecture impacts speciation across the plant and animal kingdoms. First, we briefly summarise what is known about speciation in these groups; importantly, the diversification rate of plants is about twice that of animals, and species barriers in plants may arise at an earlier stage of divergence. Next, we discuss several of the major differences in how plant and animal genomes evolve, and how they may impact the evolution of reproductive barriers and potentially speciation rates. Key differences include (1) a higher frequency of whole‐genome duplications (WGDs) and more rapid loss of synteny in plants; (2) a higher incidence and greater divergence of sex chromosomes in animals; (3) greater rates of sequence change, but slower rates of structural evolution, in animal relative to plant mitochondrial genomes; and (4) an often higher abundance of transposable elements (TEs) in plant genomes. Overall, we find the genomes of plants diverge much more rapidly in structure than those of animals (although there are many exceptions), perhaps contributing to a more rapid emergence of barriers to gene flow in plants. However, we also found that comparisons of genome evolution between the kingdoms are hampered by inconsistency in the methods employed, as well as in the metrics used to report on rates of structural evolution. Another theme from our review is the huge variation in genome architecture within each kingdom. While this variation complicates broad generalisations, it enables powerful comparative analyses that link differences in genome architecture to patterns and processes of speciation.

A FISH analysis of chromosome 17 homologs in primates suggests that genomic architecture has a direct role in karyotype evolution and in the genomic instability associated with human disease.

Plant genomics research had its beginning in December 2000, with the publication of the whole genome sequence of the model plant species Arabidopsis thaliana. Rapid progress has since been made in this area. The significant developments include the publication of a high-quality rice genome sequence in August 2005, draft genome of poplar in September 2006, whole genome sequence of two grapevine genotypes in 2007, and that of transgenic papaya in 2008. Draft sequences of corn gene-space and those of the genomes of Lotus japonicus and Glycine max have also become available in 2008. Genomes of several other plant species (e.g., Sorghum bicolor, Manihot esculenta (cassava), barley, wheat, potato, cotton, tomato, maize, Brachypodium distachyon (a small model grass genome), Medicago truncatula, shepherd's purse, peach) are also currently being sequenced. Multinational genome projects on Brassica and Solanaceous genomes are also in progress. In still other cases (e.g., wheat, corn, barley), where the large genome size prohibits whole genome sequencing, the gene rich regions (GRRs) of the genomes are being identified to bring down the sequencing work to a manageable level. The 10-year-old US National Plant Genome Initiative (NPGI) also made a call for more plant genomes to be sequenced. While making a choice for additional plant genomes to be sequenced, it has also been emphasized that much of plant diversity is available in tropical plants so that during the next decade, more genomes from tropics (e.g., Carica, Saccharum, Psychoria, Opuntia) need to be sequenced. The sequencing information obtained as above will be utilized for both basic and applied research so that while this will help in elucidating evolutionary relationships and developing better phylogenetic classification, this will also help in the discovery of new genes, allele-mining, and large-scale SNP genotyping. In order to achieve these objectives, there has also been a call for sequencing genomes of diverse cultivars of each crop like rice. As a result, the concept of plant pan genome (initially developed for microbial genomes), each composed of “core genome” and “dispensable genome,” has also been introduced. The sequence information from diverse cultivars in a crop will be utilized for molecular breeding. For instance, new technologies have been used for the improvement of indica rice, but similar efforts are now being made for improvement of japonica rice also. An overview of the present status of plant genomics research and its impact is also available in a recent special issue of Science (April 25, 2008). The future plant genomics research will certainly derive benefit from the recent development of new-generation sequencing technologies. These new technologies include improvements in sequencing systems based on Sanger's sequencing approach, as well as a number of non-Sanger sequencing technologies that became available during 2005–2008. The non-Sanger technologies include both sequencing based on amplified DNA molecules, and those based on single DNA molecules including Helicos true single molecule sequencing (tSMS) technology commercially launched in 2008. These new-generation sequencing technologies will certainly help in plant genomics research in a big way and may include a variety of research projects. While more plant genomes will be sequenced, epigenomes, transcriptomes, and metabolomes will also be worked out with much higher speed and at a cost reduced by several orders in magnitude. The science of plant genomics will also be influenced by the new emerging areas of “chemogenomics” and “synthetic genomics.” This special issue of the International Journal of Plant Genomics is devoted to “Genomics of Major Crops and Model Plant Species” with the aim to present an updated account of the genomics of major crop species and the model plant species. Articles published in this special issue involve almost all fields of genomics, including structural genomics, functional genomics, proteomics, metabolomics, and comparative genomics. Discussions also extend to cover phenomics, bioinformatics, epigenetics, and organellar genomics. Translational genomics from model plant species to cultivated crops and applications of genomics in crop improvement are topics for several articles. Structural genomics, as a major field for most crop plants, received a greater attention in this special issue, compared to other fields, including various types of molecular markers from RFLP to SNP and their use in construction of genetic, cytogenetic, and physical maps, QTL/gene mapping, genome sequencing, and generation of genomics resources. Functional genomics is the second field that received more attention, and some issues addressed significantly include gene isolation through map-based cloning and candidate gene approach, as well as functional analysis through insertional mutagenesis, RNAi, TILLING, and transcription profiling. There are 14 review articles in this special issue, seven belonging to grass family, two devoted to legumes (soybean and Medicago), one devoted to oil-seed crop (Brassica rapa), and one each to cotton, tomato, potato, and Citrus. The special issue starts with several articles on genomics of food crops including wheat, barley and rice. There is a comprehensive article on wheat genomics written by P. K. Gupta et al. (Meerut, India) followed by an article giving an overview on barley genomics by N. Sreenivasulu et al. from IPK (Gatersleben, Germany). On rice genomics, there are two articles: one with emphasis on genome sequencing (written by T. Matsumoto et al. (Japan)) gives an account of international collaboration in sequencing rice genome and its annotation (including structure and composition of rice centromeres and telomeres), and the other on rice molecular breeding (written jointly by B. Collard (Queensland, Australia) and the rice genomics group (including D. J. Mackill) from International Rice Research Institute (IRRI) (Manila, Philippines)) gives a detailed account of how rice genomics resources can be utilized for molecular breeding. A. H. Paterson has written a review on Sorghum genomics (giving information on both markers and whole genome sequencing) and H. Budak et al. (from Turkey and Spain) give an updated account of the development of genomics resources for the grass genus Brachypodium, which is being preferred over the rice genus Oryza as a model for temperate grasses (including cereals and forage grasses). G. M. Souza et al. (Brazil) have written an article on sugarcane functional genomics, outlining the development and the use of ESTs and cDNA microarrays for gene discovery. Among legume species, soybean (Glycine max) is an important crop world-wide, while Medicago truncatula and Lotus japonicus emerged as model systems for legume biology during the last decade. Therefore, one article on soybean genomics and another on Medicago truncatula have been included in this special issue. D. A. Lightfoot from The Illinois Soybean Center (Illinois, USA) discusses the use of Forresr cultivar for the development of genomic resources in this crop. Similarly, Julia Frugoli (SC, USA) with his two other colleagues elsewhere wrote an article on Medicago truncatula giving an updated account on the developments of genomic resources in this model legume. C. P. Hong et al. report the current understanding of the genome structure of Brassica rapa and efforts for the whole-genome sequencing of the species. Hong-Bin Zhang from College Station (Texas, USA) and his coworkers (from University of Georgia and China) discuss advances on genomics research in cotton, highlighting the development of DNA marker linkage/physical maps, QTL mapping, ESTs, and whole genome sequencing. The last three articles deal with genomics of two related Solanaceous crops, namely, tomato and potato, as well as a fruit-tree genus (Citrus). L. Frusciante et al. from (Portici and Roma, Italy) give an updated account of tomato genomics, G. J. Bryan and I. Hein from Scottish Crop Research Institute (SCRI, Dundee, UK) give an account of potato genomics, and M. Talon (Valencia, Spain) with F. G. Gmitter (Citrus Research and Education Center, University of Florida, USA) give an account for Citrus genomics.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.