Physical anchorage and orientation of equine linkage groups by FISH mapping BAC clones containing microsatellite markers

A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.     

FISH on avian lampbrush chromosomes produces higher resolution gene mapping

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1–6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.     

FISH mapping and sequence analysis of 87 porcine BAC clones

Ninety-one bacterial artificial chromosomes (BAC) clones, selected effectively at random from our library, were used as probes for fluorescence in situ hybridization. Of these, 87 clones gave a specific signal in one, two or three different pair(s) of swine metaphase chromosomes. The ends of 35 BAC clones were sequenced in order to obtain information for comparative mapping. Fifteen of them gave useful comparative mapping information.     

Chromosome analysis of walking catfish,Clarias magur (Teleostei,Siluriformes, Clariidae), using staining,FISH with 18S, 5S and BAC probes

The chromosomal characteristics of Clarias magur were examined using conventional (Giemsa-staining, Ag-impregnation and CMA₃ + DAPI fluorescence) and molecular/ FISH (18S & 5S rDNA probes + one BAC DNA probe) cytogenetic tools. The diploid chromosome number was 50 and the karyotype consisted of 14 metacentric, 20 sub-metacentric, 8 sub-telocentric, 8 acrocentric chromosomes with 84 chromosome arms without any heteromorphic pair. The C-heterochromatic blocks were located on centromeric position of 13 pairs of chromosomes. The NOR sites, visualized by AgNO₃- and CMA₃- staining, were situated at p arms of chromosome pair No. 21, which also corresponded to 18S rDNA site visualized by FISH. The FISH signal of ICF_001_D19 clone probe was observed on 18th chromosome pair. The findings of the present study on C. magur provided valuable markers for the chromosome identification and locations of genes of the BAC clone on the chromosome will lead to the construction of physical map of genome of this species.     

The isolation and mapping of 19 tetranucleotide microsatellite markers in the chicken

Microsatellites consisting of tetranucleotide repeats are more easily, and consequently efficiently, scored than loci consisting of dinucleotides. However, they are much less frequent in the genome. A hybridisation selection protocol was therefore employed to generate a chicken genomic library enriched for inserts containing the tetranucleotide repeat motif (TTTC)n. Forty-five new microsatellite sequences were isolated that mainly consisted of perfect repeats of the tetranucleotide (TTTC) motif. Nineteen markers were mapped in one or both of the East Lansing and Compton international chicken reference populations.     

Construction and characterisation of a BAC library from Arabidopsis halleri: Evaluation of physical mapping based on conserved synteny with Arabidopsis thaliana

We constructed and characterized the first large-insert DNA BAC library for Arabidopsis halleri, a close relative of Arabidopsis thaliana. Double size selection of high molecular weight DNA was performed to increase the average insert size. The BAC library consists of 6128 clones of which 87% have an insert size above 125 kb. Organellar DNA contamination is estimated to 1.4%. The coverage of the library is equivalent to 4.5 times the haploid genome (250 Mb), indicating the library is suitable for almost any application. We explored the possibility of generating a physical map of A. halleri using the high conserved synteny existing between this species and A. thaliana. A set of unigenes separated by 50 kb in a 850 kb region of A. thaliana chromosome II was used to probe the library. The A. halleri BAC clones isolated with these probes were grouped into two contigs. Analysis of BAC-end sequences revealed that the two A. halleri genomic contigs were highly colinear with the A. thaliana genome. Therefore, the exploitation of the conserved synteny existing between the two species will greatly facilitate the construction of a raw full physical map of A. halleri.     

Chromosome assignment of six dog genes by FISH, and correlation with dog-human Zoo-FISH data

Cross-species chromosome painting analyses have recently demonstrated the presence of regions of conserved synteny between the human and domestic dog genomes, aiding the search for candidate genes for inherited traits. Concerted efforts to subchromosomally assign substantial numbers of dog gene sequences are now needed in order to refine these comparative data, both in terms of marker density and resolution. We have developed novel PCR markers representing three dog genes (ALB, FOS, HNRPA2B1) for which no sequence or mapping data were previously available, to our knowledge. These, in addition to three gene markers previously described (ALDOA, RPE65, VCAM1), were used to isolate and chromosomally assign corresponding large insert genomic clones by fluorescence in situ hybridization (FISH). Chromosome assignments for these six dog genes are discussed in terms of those of the human orthologues, and correlated with existing comparative mapping information, identifying one apparent exception to existing Zoo-FISH data, and aiding refinement of the boundaries of conserved chromosome segments in both genomes.     

Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.     

Next‐generation sequencing,FISH mapping and synteny‐based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage‐specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian‐like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009 , Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia‐Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine‐scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.     

Radiation hybrid comparative mapping between human chromosome 17 and porcine chromosome 12 demonstrates conservation of gene order

A comparative study of human chromosome 17 (HSA17) and pig chromosome 12 (SSC12) was conducted using both somatic cell hybrid panel (SCHP) and radiation hybrid (RH) panel analysis. Sequences from an expressed sequence tag (EST) project in pig reproduction were examined and six genes and ESTs originally believed to map to HSA17 were selected for this study. The genes/ESTs were TATA box binding protein-associated factor (TAF2N/RBP56), alpha-2-plasmin inhibitor (SERPINF2/PLI), H3 histone family 3B (H3F3B), aminopeptidase puromycin sensitive (NPEPPS), an expressed sequence tag (ESTMI015) and P311 protein (P311). The SCHP analysis mapped five genes/ESTs (TAF2N, H3F3B, SERPINF2, NPEPPS and ESTMI015) to SSC12q11-q15 and SSC12p11-p15 with 100% concordance, and assigned P311 to SSC2 (1/2q24)-q29 with 100% concordance. Radiation hybrid analysis of all six genes confirmed the SCHP mapping results, with average retention frequency of 25%. Recent human sequence data demonstrated that P311 is actually located on HSA5q. As HSA5q and SSC2q show conserved syntenic regions predicted from bi-directional painting, our P311 mapping data is consistent with these results. An expanded comparative SSC12 RH map integrating the five new type I markers and 23 previously mapped loci was established using a LOD score threshold of 4.8. The gene order of the five genes/ESTs on the SSC12 framework RH map (H3F3B-ESTMI015-NPEPPS-TAF2N-SERPINF2) is identical to the HSA17 GB4 map but with inversion of the map as conventionally drawn.     

Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.     

Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.