Comparative Physical Mapping Research Articles

Genome-Wide Analysis and Expression Profile of Nuclear Factor Y (NF-Y) Gene Family in Z. jujuba.

Nuclear factor-Y (NF-Y) is an important transcription factor in the plant species, which potentially provides a higher level of functional diversity including for abiotic stress tolerance. The genome-wide study and expression analysis of NF-Y gene family in Ziziphus, an elite abiotic stress-tolerant species, assist bioprospecting of genes. Here, a total of 32 NF-Y (8 NF-YA, 15 NF-YB, and 9 NF-YC) genes were identified in genome-wide search of Z. jujuba genome. Physicochemical properties, cellular localization, gene structure, chromosomal location, and protein motifs were analyzed for structural and functional understanding. Identified 12 NF-Ys were responsible for the expansion of NF-Y gene family by tandem duplication in Z. jujuba. Phylogenetic and comparative physical mapping of Z. jujuba NF-Ys with its orthologs illustrated evolutionary and functional insights into NF-Y gene family. A total of 45 perfect microsatellites (20bp to 40bp) were extracted across the ZjNF-Y genes. The promoter and gene ontology study suggested that Z. jujuba NF-Y gene family is functionally diverse and could play a wide-ranging role in plant abiotic stress, development, and cellular processes. An expression study revealed that large numbers of the NF-Ys are differentially expressed in response to drought and salinity. The total 15 and 18 ZjNF-Y genes that are upregulated under drought and salinity stress, respectively, are the potential candidates for further functional analysis for development of climate-resilient crops. The present study established a base for understanding the role of NF-Ys in Z. jujuba under abiotic stress conditions and paved a way for further research.

Genome-wide investigation of GRAM-domain containing genes in rice reveals their role in plant-rhizobacteria interactions and abiotic stress responses

A comprehensive genome-wide survey of GRAM-domain containing genes in rice identified total 64 genes which were grouped into six classes and were physically mapped onto different rice chromosomes. GRAM domain-containing genes showed total 8 segmental and 3 tandem duplications. Comparative physical mapping between rice OsGRAM and its orthologs in related C4-crops depicted evolutionary insights into this gene family. Expression analyses of OsGRAM genes in rice roots subjected to salt stress with or without Bacillus amyloliquefaciens (SN13) inoculation revealed significant differential expression patterns suggesting their crucial role in beneficial plant-rhizobacteria interactions under stress. Further, expression analyses of selected 15 candidate genes with ≥3.0-fold induction in salt + SN13 treated samples indicated their precise and overlapping expression patterns under various abiotic stresses and phytohormones at early (1 h) and late (24 h) durations which might be ultimately responsible for functional divergence and beneficial plant-microbe interactions. Furthermore, OsGRAM27 and OsGRAM47 could be considered as potential candidate genes for further functional characterization and application in crop improvement since these genes showed positive modulation in stress under the influence of SN13. This study provides new dimensions into the evolution and divergence of OsGRAM and their role in plant-rhizobacteria interactions that could be utilized for improving stress tolerance in crops.

Partial-arm translocations in evolution of malaria mosquitoes revealed by high-coverage physical mapping of the Anopheles atroparvus genome

BackgroundMalaria mosquitoes have had a remarkable stability in the number of chromosomes in their karyotype (2n = 6) during 100 million years of evolution. Moreover, autosomal arms were assumed to maintain their integrity even if their associations with each other changed via whole-arm translocations. Here we use high-coverage comparative physical genome mapping of three Anopheles species to test the extent of evolutionary conservation of chromosomal arms in malaria mosquitoes.ResultsIn this study, we developed a physical genome map for Anopheles atroparvus, one of the dominant malaria vectors in Europe. Using fluorescence in situ hybridization (FISH) of DNA probes with the ovarian nurse cell polytene chromosomes and synteny comparison, we anchored 56 genomic scaffolds to the An. atroparvus chromosomes. The obtained physical map represents 89.6% of the An. atroparvus genome. This genome has the second highest mapping coverage among Anophelinae assemblies after An. albimanus, which has 98.2% of the genome assigned to its chromosomes. A comparison of the An. atroparvus, An. albimanus, and An. gambiae genomes identified partial-arm translocations between the autosomal arms that break down the integrity of chromosome elements in evolution affecting the structure of the genetic material in the pericentromeric regions. Unlike An. atroparvus and An. albimanus, all chromosome elements of An. gambiae are fully syntenic with chromosome elements of the putative ancestral Anopheles karyotype. We also detected nonrandom distribution of large conserved synteny blocks and confirmed a higher rate of inversion fixation in the X chromosome compared with autosomes.ConclusionsOur study demonstrates the power of physical mapping for understanding the genome evolution in malaria mosquitoes. The results indicate that syntenic relationships among chromosome elements of Anopheles species have not been fully preserved because of multiple partial-arm translocations.

Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides.

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22-24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.

Towards the bridging of molecular genetics data across Xenopus species.

BackgroundThe clawed African frog Xenopus laevis has been one of the main vertebrate models for studies in developmental biology. However, for genetic studies, Xenopus tropicalis has been the experimental model of choice because it shorter life cycle and due to a more tractable genome that does not result from genome duplication as in the case of X. laevis. Today, although still organized in a large number of scaffolds, nearly 85 % of X. tropicalis and 89 % of X. laevis genomes have been sequenced. There is expectation for a comparative physical map that can be used as a Rosetta Stone between X. laevis genetic studies and X. tropicalis genomic research.ResultsIn this work, we have mapped using coarse-grained alignment the 18 chromosomes of X. laevis, release 9.1, on the 10 reference scaffolds representing the haploid genome of X. tropicalis, release 9.0. After validating the mapping with theoretical data, and estimating reference averages of genome sequence identity, 37 to 44 % between the two species, we have carried out a synteny analysis for 2,112 orthologous genes. We found that 99.6 % of genes are in the same organization.ConclusionsTaken together, our results make possible to establish the correspondence between 62 and 65.5 % of both genomes, percentage of identity, synteny and automatic annotation of transcripts of both species, providing a new and more comprehensive tool for comparative analysis of these two species, by allowing to bridge molecular genetics data among them.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2440-9) contains supplementary material, which is available to authorized users.

Mapping 45S and 5S ribosomal genes in chromosomes of Anostomidae fish species (Ostariophysi, Characiformes) from different Amazonian water types

The fish from the family Anostomidae represent one of the most important groups of freshwater ichthyofauna from South America, with species of high economical value. The migratory characteristic of some species, through the several Amazonian environments, takes them into waters with different physico-chemical characteristics. Cytogenetic studies on the Anostomidae demonstrate that these fishes have a conserved diploid number and karyotype macrostructure. So, to verify if this conservation occurs also in the genomic level, the current study aimed at a chromosomal comparative physical mapping, using 45S and 5S rDNA, of seven species of anostomids: Leporinus fasciatus, L. agassizi, L. friderici, L. trifasciatus, Rhytiodus macrolepis, Laemolyta taeniata, and Schizodon fasciatus, collected in different Amazonian environments. The results obtained corroborate the conservation of the karyotype macrostructure. However, significant differences were found in the distribution of heterochromatin and on the pair bearing the nucleolus organizer region. The staining of 45S and 5S rDNA by FISH highlighted, for four of the seven species, more than one chromosome pair bearing the site 45S. The 5S rDNA, although present in only one chromosome pair, varied in its chromosome and karyotype position. Thus, although the Anostomidae family has a conserved chromosomic macrostructure the use of molecular techniques revealed the presence of chromosomic translocation during the evolution of these fishes.

Genome-Wide Investigation and Expression Profiling of AP2/ERF Transcription Factor Superfamily in Foxtail Millet (Setaria italica L.)

The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (∼7%) SiAP2/ERF genes were tandem repeated and 22 (∼13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.

C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses.

C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I-V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.

Global Genomic Diversity of Oryza sativa Varieties Revealed by Comparative Physical Mapping

Bacterial artificial chromosome (BAC) physical maps embedding a large number of BAC end sequences (BESs) were generated for Oryza sativa ssp. indica varieties Minghui 63 (MH63) and Zhenshan 97 (ZS97) and were compared with the genome sequences of O. sativa spp. japonica cv. Nipponbare and O. sativa ssp. indica cv. 93-11. The comparisons exhibited substantial diversities in terms of large structural variations and small substitutions and indels. Genome-wide BAC-sized and contig-sized structural variations were detected, and the shared variations were analyzed. In the expansion regions of the Nipponbare reference sequence, in comparison to the MH63 and ZS97 physical maps, as well as to the previously constructed 93-11 physical map, the amounts and types of the repeat contents, and the outputs of gene ontology analysis, were significantly different from those of the whole genome. Using the physical maps of four wild Oryza species from OMAP (http://www.omap.org) as a control, we detected many conserved and divergent regions related to the evolution process of O. sativa. Between the BESs of MH63 and ZS97 and the two reference sequences, a total of 1532 polymorphic simple sequence repeats (SSRs), 71,383 SNPs, 1767 multiple nucleotide polymorphisms, 6340 insertions, and 9137 deletions were identified. This study provides independent whole-genome resources for intra- and intersubspecies comparisons and functional genomics studies in O. sativa. Both the comparative physical maps and the GBrowse, which integrated the QTL and molecular markers from GRAMENE (http://www.gramene.org) with our physical maps and analysis results, are open to the public through our Web site (http://gresource.hzau.edu.cn/resource/resource.html).

Abstract 520: Fine Mapping of Two Loci Within a Major Albuminuria QTL on Rat Chromosome 6 That Affect Both Albuminuria and Deficient Nephron Development in The Munich Wistar Frömter Rat

We previously confirmed in the Munich Wistar Frömter (MWF) rat a functional role of a major quantitative trait locus (QTL) on rat chromosome (RNO)6 for both albuminuria development and an inherited nephron deficit in the consomic strain MWF-6SHR. We addressed the question whether an inherited nephron deficit is linked to the genetic basis of albuminuria and whether common genetic factors are responsible for both phenotypes in this rat model. We generated congenic lines to dissect the RNO6-QTL using SHR rats with low albuminuria as a reference strain. Chromosomal fine mapping was performed by breeding of subcongenic lines, comparative genome analysis and physical mapping. The nephron number was determined by determination of the glomerular density in animals at 4 weeks of age; this method was validated by comparison with the absolute nephron numbers as determined by the physical fractionator method in parental strains. Phenotyping of the congenic lines covering the RNO6-QTL demonstrated that both the albuminuria and the nephron locus were subdivided into at least two different colocalized informative loci. One interval defined by the congenic lines MWF.SHR-(D6Rat1-D6Rat81) and MWF.SHR-(D6Rat1-D6Rat117) covers 2.1 Mb and contains 26 known genes. Exchange of this locus had a strong impact on albuminuria at 8 weeks of age resulting in a decrease from 18.1 to 5.0 mg/24 h (p<0.0001) between the congenic strains and affected also the glomerular density (184 vs. 223 /mm 3 , p=0.0004). The second interval flanked by the congenic lines MWF.SHR-(D6Rat1-D6Rat117) and MWF.SHR-(D6Rat1-D6Mgh4) encompasses 2.8 Mb and contains 49 known genes. This interval influenced also albuminuria (decrease from 5.0 to 1.5 mg/24h, p=0.0001) and glomerular density (223 vs. 243 /mm 3 , p=0.008). These findings support the genetic link between albuminuria and nephron deficit in this model and provide the basis for further functional and comparative genomic analysis to obtain new insights into this association.

The X chromosome, heterochromatin and speciation in malaria mosquitoes

The available data suggest that the X chromosome of Anopheles had originated independently from the X chromosome of Drosophila. In order to gain insights into a possible role of the X chromosome in speciation of malaria mosquitoes, we studied the pattern of rearrangements, genomic landscapes and heterochromatin variations among closely and distantly related species. • Our cytogenetic analysis of the An. gambiae species complex showed fixed differences in the amount and pattern of the X heterochromatin among sibling species as well as between the M and S forms (Fig. 1). Interestingly, the low-recombination pericentric region displayed the high level of polymorphism in the size of heterochromatin and the rDNA locus (Fig. 2). • The largest region of the high genomic divergence between the incipient species (the M and S forms) co-localizes with the heterochromatin of the An. gambiae X chromosome (Fig. 3). • Our comparative physical mapping of distantly related malaria mosquito species identified the fastest rate of inversion fixation on the X chromosome despite the paucity of inversion polymorphism (Fig. 4). • The analysis of the An. gambiae genome demonstrated the highest density of transposable elements, microsatellites, minisatellites, and satellites in the X chromosome (Fig. 5). • The X chromosome is enriched in genes that may be involved in premating isolation, such as genes encoding for proteins with molecular and signal transduction activity (Fig. 6). Thus, our study points out to the elevated evolutionary plasticity of the euchromatic gene order and heterochromatin structure of the X chromosome suggesting a possible role of the sex chromosome in speciation of malaria mosquitoes. An. gambiae SUA 2La (M) An. gambiae Pimperena (S) An. gambiae Akron (M) An. gambiae Kisumu (S) Pericentric heterochromatin exhibits inter-specific differences An. merus An. quadriannulatus An. arabiensis An. gambiae Zanu (S) Fig. 1. Heterochromatin patterns observed after staining with DAPI. Arrows indicate heterochromatin (darker areas) exhibiting inter and intra-specific differences. While the M forms (Sua, Akron) exhibit single large bands (A, B), S forms (Zanu, Pimperena, Kisumu) exhibit a secondary heterochromatic band in (C-E). Sibling species (FH) also exhibit variation in heterochromatin patterns. An. gambiae Kisumu (S) An. gambiae Akron (M)

Stable karyotypes: a general rule for the fish of the family Prochilodontidae?

Cytogenetic studies involving the family Prochilodontidae have shown that these fish can be characterized by a constant diploid number and a conserved karyotypic macrostructure. This study focused on comparative physical chromosomal mapping using 18S and 5S rDNA to compare the species Semaprochilodus insignis and S. taeniurus. Our results indicated the conservation of large number of conventional chromosomal markers. The molecular cytogenetic analyses of the location of the 18S rDNA indicated the maintenance of a chromosome pair bearing these sites in both species analyzed, and it appears to be a conserved character among the majority of the species of this family. The stability of the number of 5S ribosomal DNA sites and their chromosomal localization as has been reported for the Prochilodontidae was not, however, confirmed for S. insignis and S. taeniurus, as these species showed multiple specific rDNA 5S sites. As such, and in spite of the fact that a number of studies indicate that the family Prochilodontidae has a conserved karyotypic structure, the utilization of molecular tools that use chromosomal segments as markers revealed that this presumed stability cannot be extended to the genome level for the species S. insignis and S. taeniurus.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes

BackgroundA robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species.ResultsThe turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes.ConclusionThe BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Comparative physical mapping between wheat chromosome arm 2BL and rice chromosome 4

Physical maps of chromosomes provide a framework for organizing and integrating diverse genetic information. DNA microarrays are a valuable technique for physical mapping and can also be used to facilitate the discovery of single feature polymorphisms (SFPs). Wheat chromosome arm 2BL was physically mapped using a Wheat Genome Array onto near-isogenic lines (NILs) with the aid of wheat-rice synteny and mapped wheat EST information. Using high variance probe set (HVP) analysis, 314 HVPs constituting genes present on 2BL were identified. The 314 HVPs were grouped into 3 categories: HVPs that match only rice chromosome 4 (298 HVPs), those that match only wheat ESTs mapped on 2BL (1), and those that match both rice chromosome 4 and wheat ESTs mapped on 2BL (15). All HVPs were converted into gene sets, which represented either unique rice gene models or mapped wheat ESTs that matched identified HVPs. Comparative physical maps were constructed for 16 wheat gene sets and 271 rice gene sets. Of the 271 rice gene sets, 257 were mapped to the 18-35Mb regions on rice chromosome 4. Based on HVP analysis and sequence similarity between the gene models in the rice chromosomes and mapped wheat ESTs, the outermost rice gene model that limits the translocation breakpoint to orthologous regions was identified.

Comparative physical maps derived from BAC end sequences of tilapia (Oreochromis niloticus)

BackgroundThe Nile tilapia is the second most important fish in aquaculture. It is an excellent laboratory model, and is closely related to the African lake cichlids famous for their rapid rates of speciation. A suite of genomic resources has been developed for this species, including genetic maps and ESTs. Here we analyze BAC end-sequences to develop comparative physical maps, and estimate the number of genome rearrangements, between tilapia and other model fish species.ResultsWe obtained sequence from one or both ends of 106,259 tilapia BACs. BLAST analysis against the genome assemblies of stickleback, medaka and pufferfish allowed identification of homologies for approximately 25,000 BACs for each species. We calculate that rearrangement breakpoints between tilapia and these species occur about every 3 Mb across the genome. Analysis of 35,000 clones previously assembled into contigs by restriction fingerprints allowed identification of longer-range syntenies.ConclusionsOur data suggest that chromosomal evolution in recent teleosts is dominated by alternate loss of gene duplicates, and by intra-chromosomal rearrangements (~one per million years). These physical maps are a useful resource for comparative positional cloning of traits in cichlid fishes. The paired BAC end sequences from these clones will be an important resource for scaffolding forthcoming shotgun sequence assemblies of the tilapia genome.

Cytogenetic map for Anopheles nili: Application for population genetics and comparative physical mapping

Anopheles nili is one of the major malaria vectors in Africa with a wide geographic distribution. However, the taxonomic and population genetic studies on this species are scarce. New research tools are urgently needed to genetically characterize this important malaria vector. In this study, a high-resolution cytogenetic map was developed for An. nili polytene chromosomes. Chromosomes were straightened and subdivided into 46 numbered divisions according to the banding pattern. Population analysis of An. nili females collected in Burkina Faso revealed the presence of two highly polymorphic inversions on the 2R chromosomal arm. A statistically significant departure from Hardy–Weinberg equilibrium due to a deficit in heterozygotes was detected for inversion 2R b. To determine chromosome homologies and gene order conservation between An. nili and other major malaria vectors, PCR probes based on the An. gambiae coding sequences were mapped to An. nili chromosomes. Comparative mapping demonstrated that An. nili chromosomes have an An. stephensi-like arm association and that whole-arm translocations and paracentric inversions were the major types of rearrangement in evolution of these mosquitoes. The minimum number of fixed inversions among An. nili, An. gambiae, and An. stephensi was calculated using the Multiple Genome Rearrangements (MGR), Genome Rearrangements In Man and Mouse (GRIMM), and Sorting Permutation by Reversals and block-INterchanGes (SPRING) programs. The data suggest that the An. nili is, at least, as diverged from An. gambiae as An. stephensi. We provide evidence that 2L a/ a arrangement of An. gambiae is present in outgroup species An. nili and An. stephensi confirming the ancestral status of the 2L a inversion in the An. gambiae complex. Availability of the new polytene chromosome map, polymorphic inversions, and physically mapped DNA markers for An. nili will further stimulate population genetic, taxonomic, and genomic studies of this neglected malaria vector.

Microsatellites inBrassicaunigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis

Microsatellites present in the transcribed regions of the genome have the potential to reveal functional diversity. Unigene sequence databases are the sources of such genic microsatellites with unique flanking sequences and genomic locations even in complex polyploids. The present study was designed to assay the unigenes of Brassica napus and B. rapa for various microsatellite repeats, and to design markers and use them in comparative genome analysis and study of evolution. The average frequency of microsatellites in Brassica unigenes was one in every 7.25 kb of sequence, as compared with one in every 8.57 kb of sequence in Arabidopsis thaliana. Trinucleotide motifs coding for serine and the dinucleotide motif GA were most abundant. We designed 2374 and 347 unigene-based microsatellite (UGMS) markers including 541 and 58 class I types in B. napus and B. rapa, respectively, and evaluated their use across diverse species and genera. Most of these markers (93.3%) gave successful amplification of target microsatellite motifs, which was confirmed by sequencing. Interspecific polymorphism between B. napus and B. rapa detected in silico for the UGMS markers was 4.16 times higher in 5' untranslated regions than in coding sequences. Physical anchoring of Brassica UGMS markers on the A. thaliana genome indicated their significance in studying the evolutionary history of A. thaliana genomic duplications in relation to speciation. Comparative physical mapping identified 85% of Brassica unigenes as single copy and gave clues for the presence of conserved primordial gene order. Complex chromosomal rearrangements such as inversions, tandem and segmental duplications, and insertions/deletions were evident between A. thaliana and B. rapa genomes. The results obtained have encouraging implications for the use of UGMS markers in comparative genome analysis and for understanding evolutionary complexities in the family Brassicaceae.

Genic non-coding microsatellites in the rice genome: characterization, marker design and use in assessing genetic and evolutionary relationships among domesticated groups

BackgroundCompletely sequenced plant genomes provide scope for designing a large number of microsatellite markers, which are useful in various aspects of crop breeding and genetic analysis. With the objective of developing genic but non-coding microsatellite (GNMS) markers for the rice (Oryza sativa L.) genome, we characterized the frequency and relative distribution of microsatellite repeat-motifs in 18,935 predicted protein coding genes including 14,308 putative promoter sequences.ResultsWe identified 19,555 perfect GNMS repeats with densities ranging from 306.7/Mb in chromosome 1 to 450/Mb in chromosome 12 with an average of 357.5 GNMS per Mb. The average microsatellite density was maximum in the 5' untranslated regions (UTRs) followed by those in introns, promoters, 3'UTRs and minimum in the coding sequences (CDS). Primers were designed for 17,966 (92%) GNMS repeats, including 4,288 (94%) hypervariable class I types, which were bin-mapped on the rice genome. The GNMS markers were most polymorphic in the intronic region (73.3%) followed by markers in the promoter region (53.3%) and least in the CDS (26.6%). The robust polymerase chain reaction (PCR) amplification efficiency and high polymorphic potential of GNMS markers over genic coding and random genomic microsatellite markers suggest their immediate use in efficient genotyping applications in rice. A set of these markers could assess genetic diversity and establish phylogenetic relationships among domesticated rice cultivar groups. We also demonstrated the usefulness of orthologous and paralogous conserved non-coding microsatellite (CNMS) markers, identified in the putative rice promoter sequences, for comparative physical mapping and understanding of evolutionary and gene regulatory complexities among rice and other members of the grass family. The divergence between long-grained aromatics and subspecies japonica was estimated to be more recent (0.004 Mya) compared to short-grained aromatics from japonica (0.006 Mya) and long-grained aromatics from subspecies indica (0.014 Mya).ConclusionOur analyses showed that GNMS markers with their high polymorphic potential would be preferred candidate functional markers in various marker-based applications in rice genetics, genomics and breeding. The CNMS markers provided encouraging implications for their use in comparative genome mapping and understanding of evolutionary complexities in rice and other members of grass family.

Uprobe 2008: an online resource for universal overgo hybridization-based probe retrieval and design

Cross-species sequence comparisons are a prominent method for analyzing genomic DNA and an ever increasing number of species are being selected for whole-genome sequencing. Targeted comparative genomic sequencing is a complementary approach to whole-genome shotgun sequencing and can produce high-quality sequence assemblies of orthologous chromosomal regions of interest from multiple species. Genomic libraries necessary to support targeted mapping and sequencing projects are available for more than 90 vertebrates. An essential step for utilizing these and other genomic libraries for targeted mapping and sequencing is the development of the hybridization-based probes, which are necessary to screen a genomic library of interest. The Uprobe website (http://uprobe.genetics.emory.edu) provides a public online resource for identifying or designing ‘universal’ overgo-hybridization probes from conserved sequences that can be used to efficiently screen one or more genomic libraries from a designated group of species. Currently, Uprobe provides the ability to search or design probes for use in broad groups of species, including mammals and reptiles, as well as more specific clades, including marsupials, carnivores, rodents and nonhuman primates. In addition, Uprobe has the capability to design custom probes from multiple-species sequence alignments provided by the user, thus providing a general tool for targeted comparative physical mapping.

Construction of a California condor BAC library and first-generation chicken–condor comparative physical map as an endangered species conservation genomics resource

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing ∼14-fold coverage of the presumed ∼1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken–condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8× subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken–condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.