Libraries of large-insert genomic clones as a tool for molecular cytogenetic analysis of avian genome

Integration of molecular and cytegenetic levels of investigation results in complex understanding of structural and functional genome organization. Gridded libraries of large-insert genomic clones represent a powerful tool of the genome analysis. Their utilization provides coordination of data on molecular organization of nucleic acids with cytogenetic data on the chromosome structure. These libraries played an important role in sequencing of genomes of human, mouse, and other organisms as an instrument linking molecular biological and cytogenetic data via construction of contigs and their localization on the chromosomes. They also enabled analysis of orthology between the mammalian genomes. The existing avian libraries fit molecular cytogenetic analysis of the class Aves genome, and can be successfully used for the isolation and characterization of large genomic fragments. This provides utilization of these libraries not only for the chromosome mapping, but also for positional cloning and search for candidate genes for quantitative traits.     

Comparative cytogenetics of rodents

Here, we present analysis of data on comparative chromosome painting produced using various chromosome-specific libraries for members of different Glires groups. Based on the results of comparative cytogenetic and molecular studies, the modern rodents can be conventionally classified into two groups with sharply differing directions and tempos of karyotypic evolution. One group (suborders Sciuromorpha, Castorimorpha, and Anomaluromorpha) preserved conserved genomes, which are probably close in structure to the genome of the ancestor of all mammals. The genomes of the other group (suborder Myomorpha) underwent “catastrophic evolution,” which resulted in numerous breaks and fusions of the ancient chromosomes. The current data do not allow unambiguously assigning the order Hystricomorpha to any of these groups.     

Integrating cytogenetics and genomics in comparative evolutionary studies of cichlid fish

ABSTRACT: BACKGROUND: The availability of a large number of recently sequenced vertebrate genomes opens new avenues to integrate cytogenetics and genomics in comparative and evolutionary studies. Cytogenetic mapping can offer alternative means to identify conserved synteny shared by distinct genomes and also to define genome regions that are still not fine characterized even after wide-ranging nucleotide sequence efforts. An efficient way to perform comparative cytogenetic mapping is based on BAC clones mapping by fluorescence in situ hybridization. In this report, to address the knowledge gap on the genome evolution in cichlid fishes, BAC clones of an Oreochromis niloticus library covering the linkage groups (LG) 1, 3, 5, and 7 were mapped onto the chromosomes of 9 African cichlid species. The cytogenetic mapping data were also integrated with BAC-end sequences information of O. niloticus and comparatively analyzed against the genome of other fish species and vertebrates. RESULTS: The location of BACs from LG1, 3, 5, and 7 revealed a strong chromosomal conservation among the analyzed cichlid species genomes, which evidenced a synteny of the markers of each LG. Comparative in silico analysis also identified large genomic blocks that were conserved in distantly related fish groups and also in other vertebrates. CONCLUSIONS: Although it has been suggested that fishes contain plastic genomes with high rates of chromosomal rearrangements and probably low rates of synteny conservation, our results evidence that large syntenic chromosome segments have been maintained conserved during evolution, at least for the considered markers. Additionally, our current cytogenetic mapping efforts integrated with genomic approaches conduct to a new perspective to address important questions involving chromosome evolution in fishes.     

Rice genome organization: the centromere and genome interactions

Over the last decade, many varied resources have become available for genome studies in rice. These resources include over 4000 DNA markers, several bacterial artificial chromosome (BAC) libraries, P-1 derived artificial chromosome (PAC) libraries and yeast artificial chromosome (YAC) libraries (genomic DNA clones, filters and end-sequences), retrotransposon tagged lines, and many chemical and irradiated mutant lines. Based on these, high-density genetic maps, cereal comparative maps, YAC and BAC physical maps, and quantitative trait loci (QTL) maps have been constructed, and 93 % of the genome has also been sequenced. These data have revealed key features of the genetic and physical structure of the rice genome and of the evolution of cereal chromosomes. This Botanical Briefing examines aspects of how the rice genome is organized structurally, functionally and evolutionarily. Emphasis is placed on the rice centromere, which is composed of long arrays of centromere-specific repetitive sequences. Differences and similarities amongst various cereal centromeres are detailed. These indicate essential features of centromere function. Another view of various kinds of interactive relationships within and between genomes, which could play crucial roles in genome organization and evolution, is also introduced. Constructed genetic and physical maps indicate duplication of chromosomal segments and spatial association between specific chromosome regions. A genome-wide survey of interactive genetic loci has identified various reproductive barriers that may drive speciation of the rice genome. The significance of these findings in genome organization and evolution is discussed.     

Evaluating the genomic and sequence integrity of human ES cell lines; comparison to normal genomes

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count, and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation, using both microarray and sequence-based analyses, provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines, the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly, genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes, such as tumor suppressors and genetic disease genes, do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens, such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents.     

Identification of the ``a' Genome of Finger Millet Using Chloroplast DNA

Finger millet (Eleusine corocana subsp. coracana), an important cereal in East Africa and India, is a tetraploid species with unknown genomic components. A recent cytogenetic study confirmed the direct origin of this millet from the tetraploid E. coracana subsp. africana but questioned Eleusine indica as a genomic donor. Chloroplast (ct) DNA sequence analysis using restriction fragment pattern was used to examine the phylogenetic relationships between E. coracana subsp. coracana (domesticated finger millet), E. coracana subspecies africana (wild finger millet), and E. indica. Eleusine tristachya was included since it is the only other annual diploid species in the genus with a basic chromosome number of x = 9 like finger millet. Eight of the ten restriction endonucleases used had 16 to over 30 restriction sites per genome and were informative. E. coracana subsp. coracana and subsp. africana and E. indica were identical in all the restriction sites surveyed, while the ct genome of E. tristachya differed consistently by at least one mutational event for each restriction enzyme surveyed. This random survey of the ct genomes of these species points out E. indica as one of the genome donors (maternal genome donor) of domesticated finger millet contrary to a previous cytogenetic study. The data also substantiate E. coracana subsp. africana as the progenitor of domesticated finger millet. The disparity between the cytogenetic and the molecular approaches is discussed in light of the problems associated with chromosome pairing and polyploidy.     

Towards the era of comparative evolutionary genomics in Brassicaceae

The vast genetic diversity, specific genome organization and sequencing of the Arabidopsis thaliana genome made crucifers an ideal group for comparative genomic studies. Arabidopsis genomic resources have greatly expedited comparative genomics within Brassicaceae and fostered the establishment of new Arabidopsis relative model systems (ARMS). The extent of genome colinearity, modes and evolutionary rates of genome alterations are being analyzed by genetic mapping with ever increasing levels of precision. Comparative cytogenetic studies in Brassicaceae are employing various chromosome landmarks and cytogenetic techniques, including localization of rDNA, variation in centromeric satellite repeats, genomic in situ hybridization (GISH), fluorescence ISH using bacterial artificial chromosomes (BAC FISH), and large-scale comparative chromosome painting. Some genome alterations may represent rare genomic changes (RGCs) and thus have the potential to resolve complex/conflicting phylogenetic relationships inferred from DNA sequencing. Comparative genomics should increasingly be integrated with molecular phylogenetics and population genetics to elucidate the processes responsible for genetic variation in Brassicaceae.     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

Bridging the resolution gap in structural modeling of 3D genome organization

Over the last decade, and especially after the advent of fluorescent in situ hybridization imaging and chromosome conformation capture methods, the availability of experimental data on genome three-dimensional organization has dramatically increased. We now have access to unprecedented details of how genomes organize within the interphase nucleus. Development of new computational approaches to leverage this data has already resulted in the first three-dimensional structures of genomic domains and genomes. Such approaches expand our knowledge of the chromatin folding principles, which has been classically studied using polymer physics and molecular simulations. Our outlook describes computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains.     

Telomeres,telomerase, and stability of the plant genome

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.     

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.     

Characterising chromosome rearrangements: recent technical advances in molecular cytogenetics

Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes.     

DNA, chromosomes, and in situ hybridization.

In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding.     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome （BAC） clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza sativa L. （AA genome） when used as fluorescence in situ hybridization （FISH） probes. We further probed those markers to the pachytene chromosomes of O. punctata （BB genome） and O. officinalis （CC genome） and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.     

Three Infectious Viral Species Lying in Wait in the Banana Genome

Plant pararetroviruses integrate serendipitously into their host genomes. The banana genome harbors integrated copies of banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious pararetrovirus. In this investigation, we characterized integrants of three BSV species—Goldfinger (eBSGFV), Imove (eBSImV), and Obino l''Ewai (eBSOLV)—in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape, and infectious capacity. All eBSVs exhibit extensive viral genome duplications and rearrangements. eBSV segregation analysis on an F1 population of PKW combined with fluorescent in situ hybridization analysis showed that eBSImV, eBSOLV, and eBSGFV are each present at a single locus. eBSOLV and eBSGFV contain two distinct alleles, whereas eBSImV has two structurally identical alleles. Genotyping of both eBSV and viral particles expressed in the progeny demonstrated that only one allele for each species is infectious. The infectious allele of eBSImV could not be identified since the two alleles are identical. Finally, we demonstrate that eBSGFV and eBSOLV are located on chromosome 1 and eBSImV is located on chromosome 2 of the reference Musa genome published recently. The structure and evolution of eBSVs suggest sequential integration into the plant genome, and haplotype divergence analysis confirms that the three loci display differential evolution. Based on our data, we propose a model for BSV integration and eBSV evolution in the Musa balbisiana genome. The mutual benefits of this unique host-pathogen association are also discussed.     

Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe--a landmark approach for Thinopyrum genome research

The introduction of alien genetic variation from the genus Thinopyrum through chromosome engineering into wheat is a valuable and proven technique for wheat improvement. A number of economically important traits have been transferred into wheat as single genes, chromosome arms or entire chromosomes. Successful transfers can be greatly assisted by the precise identification of alien chromatin in the recipient progenies. Chromosome identification and characterization are useful for genetic manipulation and transfer in wheat breeding following chromosome engineering. Genomic in situ hybridization (GISH) using an S genomic DNA probe from the diploid species Pseudoroegneria has proven to be a powerful diagnostic cytogenetic tool for monitoring the transfer of many promising agronomic traits from Thinopyrum. This specific S genomic probe not only allows the direct determination of the chromosome composition in wheat-Thinopyrum hybrids, but also can separate the Th. intermedium chromosomes into the J, J(S) and S genomes. The J(S) genome, which consists of a modified J genome chromosome distinguished by S genomic sequences of Pseudoroegneria near the centromere and telomere, carries many disease and mite resistance genes. Utilization of this S genomic probe leads to a better understanding of genomic affinities between Thinopyrum and wheat, and provides a molecular cytogenetic marker for monitoring the transfer of alien Thinopyrum agronomic traits into wheat recipient lines.