Transcription from a gene desert in a melanoma porcine model.

The genetic mechanisms underlying cutaneous melanoma onset and progression need to be further understood to improve patients' care. Several studies have focused on the genetic determinism of melanoma development in the MeLiM pig, a biomedical model of cutaneous melanoma. The objective of this study was to better describe the influence of a particular genomic region on melanoma progression in the MeliM model. Indeed, a large region of the Sus scrofa chromosome 1 has been identified by linkage and association analyses, but the causal mechanisms have remained elusive. To deepen the analysis of this candidate region, a dedicated SNP panel was used to fine map the locus, downsizing the interval to less than 2 Mb, in a genomic region located within a large gene desert. Transcription from this locus was addressed using a tiling array strategy and further validated by RT-PCR in a large panel of tissues. Overall, the gene desert showed an extensive transcriptional landscape, notably dominated by repeated element transcription in tumor and fetal tissues. The transcription of LINE-1 and PERVs has been confirmed in skin and tumor samples from MeLiM pigs. In conclusion, although this study still does not identify a candidate mutation for melanoma occurrence or progression, it highlights a potential role of repeated element transcriptional activity in the MeLiM model.

Towards the quantitative characterisation of piglets' robustness to weaning: a modelling approach.

Weaning is a critical transition phase in swine production in which piglets must cope with different stressors that may affect their health. During this period, the prophylactic use of antibiotics is still frequent to limit piglet morbidity, which raises both economic and public health concerns such as the appearance of antimicrobial-resistant microbes. With the interest of developing tools for assisting health and management decisions around weaning, it is key to provide robustness indexes that inform on the animals' capacity to endure the challenges associated with weaning. This work aimed at developing a modelling approach for facilitating the quantification of piglet resilience to weaning. A total of 325 Large White pigs weaned at 28 days of age were monitored and further housed and fed conventionally during the post-weaning period without antibiotic administration. Body weight and diarrhoea scores were recorded before and after weaning, and blood was sampled at weaning and 1 week later for collecting haematological data. A dynamic model was constructed based on the Gompertz-Makeham law to describe live weight trajectories during the first 75 days after weaning, following the rationale that the animal response is partitioned in two time windows (a perturbation and a recovery window). Model calibration was performed for each animal. Our results show that the transition time between the two time windows, as well as the weight trajectories are characteristic for each individual. The model captured the weight dynamics of animals at different degrees of perturbation, with an average coefficient of determination of 0.99, and a concordance correlation coefficient of 0.99. The utility of the model is that it provides biologically meaningful parameters that inform on the amplitude and length of perturbation, and the rate of animal recovery. Our rationale is that the dynamics of weight inform on the capability of the animal to cope with the weaning disturbance. Indeed, there were significant correlations between model parameters and individual diarrhoea scores and haematological traits. Overall, the parameters of our model can be useful for constructing weaning robustness indexes by using exclusively the growth curves. We foresee that this modelling approach will provide a step forward in the quantitative characterisation of robustness.

Immunome differences between porcine ileal and jejunal Peyer's patches revealed by global transcriptome sequencing of gut-associated lymphoid tissues.

The epithelium of the intestinal mucosa and the gut-associated lymphoid tissues (GALT) constitute an essential physical and immunological barrier against pathogens. In order to study the specificities of the GALT transcriptome in pigs, we compared the transcriptome profiles of jejunal and ileal Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and peripheral blood (PB) of four male piglets by RNA-Seq. We identified 1,103 differentially expressed (DE) genes between ileal PPs (IPPs) and jejunal PPs (JPPs), and six times more DE genes between PPs and MLNs. The master regulator genes FOXP3, GATA3, STAT4, TBX21 and RORC were less expressed in IPPs compared to JPPs, whereas the transcription factor BCL6 was found more expressed in IPPs. In comparison between IPPs and JPPs, our analyses revealed predominant differential expression related to the differentiation of T cells into Th1, Th2, Th17 and iTreg in JPPs. Our results were consistent with previous reports regarding a higher T/B cells ratio in JPPs compared to IPPs. We found antisense transcription for respectively 24%, 22% and 14% of the transcripts detected in MLNs, PPs and PB, and significant positive correlations between PB and GALT transcriptomes. Allele-specific expression analyses revealed both shared and tissue-specific cis-genetic control of gene expression.

Deciphering the genetic regulation of peripheral blood transcriptome in pigs through expression genome-wide association study and allele-specific expression analysis.

BACKGROUND: Efforts to improve sustainability in livestock production systems have focused on two objectives: investigating the genetic control of immune function as it pertains to robustness and disease resistance, and finding predictive markers for use in breeding programs. In this context, the peripheral blood transcriptome represents an important source of biological information about an individual's health and immunological status, and has been proposed for use as an intermediate phenotype to measure immune capacity. The objective of this work was to study the genetic architecture of variation in gene expression in the blood of healthy young pigs using two approaches: an expression genome-wide association study (eGWAS) and allele-specific expression (ASE) analysis. RESULTS: The blood transcriptomes of 60-day-old Large White pigs were analyzed by expression microarrays for eGWAS (242 animals) and by RNA-Seq for ASE analysis (38 animals). Using eGWAS, the expression levels of 1901 genes were found to be associated with expression quantitative trait loci (eQTLs). We recovered 2839 local and 1752 distant associations (Single Nucleotide Polymorphism or SNP located less or more than 1 Mb from expression probe, respectively). ASE analyses confirmed the extensive cis-regulation of gene transcription in blood, and revealed allelic imbalance in 2286 SNPs, which affected 763 genes. eQTLs and ASE-genes were widely distributed on all chromosomes. By analyzing mutually overlapping eGWAS results, we were able to describe putative regulatory networks, which were further refined using ASE data. At the functional level, genes with genetically controlled expression that were detected by eGWAS and/or ASE analyses were significantly enriched in biological processes related to RNA processing and immune function. Indeed, numerous distant and local regulatory relationships were detected within the major histocompatibility complex region on chromosome 7, revealing ASE for most class I and II genes. CONCLUSIONS: This study represents, to the best of our knowledge, the first genome-wide map of the genetic control of gene expression in porcine peripheral blood. These results represent an interesting resource for the identification of genetic markers and blood biomarkers associated with variations in immunity traits in pigs, as well as any other complex traits for which blood is an appropriate surrogate tissue.

Review: divergent selection for residual feed intake in the growing pig.

This review summarizes the results from the INRA (Institut National de la Recherche Agronomique) divergent selection experiment on residual feed intake (RFI) in growing Large White pigs during nine generations of selection. It discusses the remaining challenges and perspectives for the improvement of feed efficiency in growing pigs. The impacts on growing pigs raised under standard conditions and in alternative situations such as heat stress, inflammatory challenges or lactation have been studied. After nine generations of selection, the divergent selection for RFI led to highly significant (P<0.001) line differences for RFI (-165 g/day in the low RFI (LRFI) line compared with high RFI line) and daily feed intake (-270 g/day). Low responses were observed on growth rate (-12.8 g/day, P<0.05) and body composition (+0.9 mm backfat thickness, P=0.57; -2.64% lean meat content, P<0.001) with a marked response on feed conversion ratio (-0.32 kg feed/kg gain, P<0.001). Reduced ultimate pH and increased lightness of the meat (P<0.001) were observed in LRFI pigs with minor impact on the sensory quality of the meat. These changes in meat quality were associated with changes of the muscular energy metabolism. Reduced maintenance energy requirements (-10% after five generations of selection) and activity (-21% of time standing after six generations of selection) of LRFI pigs greatly contributed to the gain in energy efficiency. However, the impact of selection for RFI on the protein metabolism of the pig remains unclear. Digestibility of energy and nutrients was not affected by selection, neither for pigs fed conventional diets nor for pigs fed high-fibre diets. A significant improvement of digestive efficiency could likely be achieved by selecting pigs on fibre diets. No convincing genetic or blood biomarker has been identified for explaining the differences in RFI, suggesting that pigs have various ways to achieve an efficient use of feed. No deleterious impact of the selection on the sow reproduction performance was observed. The resource allocation theory states that low RFI may reduce the ability to cope with stressors, via the reduction of a buffer compartment dedicated to responses to stress. None of the experiments focussed on the response of pigs to stress or challenges could confirm this theory. Understanding the relationships between RFI and responses to stress and energy demanding processes, as such immunity and lactation, remains a major challenge for a better understanding of the underlying biological mechanisms of the trait and to reconcile the experimental results with the resource allocation theory.

Early modulation of the cecal microbial activity in the young rabbit with rapidly fermentable fiber: impact on health and growth.

This study aimed at comparing various diets predicted to induce different stimulations of the cecal microbial activity of the young rabbit fed ad libitum from 16 to 70 d of age: i) a diet enriched with rapidly fermentable fiber expected to stimulate the cecal microbial activity (RFF group); ii) a control diet with a standard composition (C group); iii) and the same control diet with tiamulin and apramycin antibiotics, expected to inhibit the microbial activity (C+AB group). A total of 398 rabbits were used from 42 litters and weaned at 28 d of age. An in vivo digestibility trial was performed on 36 rabbits of 42 to 46 d of age housed in individual metabolic cages. The feed intake and growth rates were lower in the RFF group compared with the C+AB group (-15% in ADFI and -11% in ADG, P<0.001), with a lower weight of -183 g at 70 d (P<0.001). No significant difference was found on ADG and final BW between the RFF and the C groups, but the RFF diet allowed a better G:F ratio at postweaning (P<0.01). The digestion of soluble fiber (total dietary fiber minus NDF) was greater for the RFF group. The C+AB diet had a positive effect on the postweaning morbidity rate (P<0.05) but did not affect the mortality rate and the health risk index (morbidity and mortality). Conversely, the RFF diet appeared to reduce the mortality rate compared with the C+AB diet, especially before 41 d of age. Concerning the cecal microbial activity, a supply of RFF in the diet increased the cecal VFA concentrations (+28% vs. C+AB and +22% vs. C, P<0.001) and lowered the pH. The VFA pattern was affected at 45 and 60 d, with a dominance of acetate in the RFF group (+4% vs. C+AB and C groups, P<0.001) instead of butyrate in the C+AB and C groups (-3.6% and -5% vs. C+AB and C, respectively, P<0.001). Antibiotics addition (C+AB group) reduced the VFA concentration, but only after weaning (-25% at 45 d of age) without changing the fermentation pattern. In conclusion, early intake of RFF in young rabbits stimulated the cecal microbial activity, and reduced the voluntary feed intake, leading to a reduced G:F ratio.

Exploring copy number variation in the rabbit (Oryctolagus cuniculus) genome by array comparative genome hybridization.

The European rabbit (Oryctolagus cuniculus) is relevant in a large spectrum of fields: it is a livestock, a pet, a biomedical model and a biotechnology tool, a wild resource and a pest. The sequencing of the rabbit genome has opened new perspectives to study this lagomorph at the genome level. We herein investigated for the first time the O. cuniculus genome by array comparative genome hybridization (aCGH) and established a first copy number variation (CNV) genome map in this species comprising 155 copy number variation regions (CNVRs; 95 gains, 59 losses, 1 with both gain and loss) covering ~0.3% of the OryCun2.0 version. About 50% of the 155 CNVRs identified spanned 139 different protein coding genes, 110 genes of which were annotated or partially annotated (including Major Histocompatibility Complex genes) with 277 different gene ontology terms. Many rabbit CNVRs might have a functional relevance that should be further investigated.

Transcription specificity of the class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex and comparison with class Ia genes.

Our aim was to analyse the transcription levels of the three non-classical class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex in various tissues and conditions and to compare them to the transcription levels of classical class Ia genes. Twenty-five adult tissues from two pig breeds, pig renal PK15 cells infected with the Pseudorabies virus, and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide or a mixture of phorbol myristate acetate and ionomycin were included in our study. Relative transcription was quantified by quantitative real-time PCR. On average, in adult tissues and PBMCs and compared to SLA-6, the transcription level of SLA-Ia genes was 100-1000 times higher, the level of SLA-8 was 10-20 times higher, and that of SLA-7 was five times higher. Thus, SLA-8 is the most transcribed SLA-Ib gene, followed by the SLA-7 and SLA-6 genes. The highest transcription levels of SLA-Ib transcripts were found in the lymphoid organs, followed by the lung and the digestive tract. The tissue variability of expression levels was widest for the SLA-6 gene, with a 1:32 ratio between the lowest and highest levels in contrast to a 1:12 ratio for the SLA-7 and SLA-8 genes and a 1:16 ratio for the SLA-Ia genes. During PK-15 infection and PBMC stimulation, SLA-Ia and SLA-8 genes were downregulated, whereas SLA-6 and SLA-7 were upregulated, downregulated or not significantly modified. Our overall results confirm the tissue-wide transcription of the three SLA-Ib genes and suggest that they have complementary roles.

Effect of polyethylene glycol-based preservation solutions on graft injury in experimental kidney transplantation.

BACKGROUND: New preservation solutions are emerging, of various ionic compositions and with hydroxyethyl starch replaced by polymers such as polyethylene glycols (PEGs), offering the potential for 'immunocamouflage'. This experimental study investigated which of three clinically available preservation protocols offered the best graft protection, based on epithelial-to-mesenchymal transition (EMT) and fibrosis. METHODS: Kidneys were preserved for 24 h at 4° C with University of Wisconsin solution (UW)as standard, compared with solutions containing either 1 g/l PEG 35 kDa (Institute Georges Lopez solution, IGL) or 30 g/l PEG 20 kDa (solution de conservation des organes et des tissus, SCOT). Animals were followed for up to 3 months and development of EMT, tubular atrophy and fibrosis was evaluated in comparison with sham-operated animals. RESULTS: Functional recovery was better in the SCOT group compared with the other groups. Chronic fibrosis, EMT and inflammation were observed in the UW and IGL groups, but limited in the SCOT group. Levels of profibrosis markers such as transforming growth factor ß1, plasminogen activator inhibitor 1 and connective tissue growth factor were increased in IGL and UW groups compared with the SCOT group. Hypoxia-inducible factor (HIF) 1α and 2α expression was increased at 3 months in grafts preserved in UW and IGL, but detected transiently on day 14 when SCOT was used. Expression of HIF-regulated genes vascular endothelial growth factor and erythropoietin was increased in UW and IGL groups. CONCLUSION: The choice of colloid and ionic content is paramount in providing long-term protection against chronic graft injury after renal transplantation. Preservation solutions based on PEGs may optimize graft quality.

Nomenclature for factors of the SLA system, update 2008.

This report summarizes the new swine leukocyte antigen (SLA) allele sequences and haplotypes designated by the SLA Nomenclature Committee of the International Society for Animal Genetics. There have been 74 new SLA alleles, comprising 18 SLA-1 alleles, 11 SLA-2 alleles, six SLA-3 alleles, two SLA-6 alleles, one SLA-DRA allele, 20 SLA-DRB1 alleles, three SLA-DQA alleles and 13 SLA-DQB1 alleles. Twelve new SLA class I and four new class II haplotypes have also been designated. This is the first official update since the 2005 reports on the nomenclature for factors of the SLA class I and II systems. This report also summarizes recent updates to the Immunopolymorphism Database-Major Histocompatibility Complex (IPD-MHC) website (http://www.ebi.ac.uk/ipd/mhc/sla/). All information has now been integrated to the SLA section of the IPD-MHC database, which serves as the repository for maintaining a list of all recognized SLA genes and their allelic sequences.

A combined transcriptomic approach to analyse the dialogue between pseudorabies virus and porcine cells.

The pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for the study of virus-host cell dialog. As PrV has a strong tropism for mucous epithelial cells, we chose to follow in vitro the PrV time course-infection of porcine PK15 cells. The viral and cellular transcriptome modifications were simultaneously analysed using a combined SLA/PrV cDNA microarray, the porcine Qiagen-NRSP8 oligonucleotides microarray and real time quantitative PCR.Ahigh increase in viral gene expression was found from 4 h post-infection (PI), concomitantly to the first viral progeny and most viral genes were differentially expressed 12 h PI. No early global cellular shutoff was observed but many cellular genes were downregulated between 8 and 12 h PI, when UL41 transcripts encoding the virion shutoff protein, were first detected. Several genes involved in the MHC class I mediated antigenic pathway were downregulated including SLA-la, TAP1, TAP2, PSMB8 and PSMB9 genes. These results suggested that PrV prevents the viral antigen presentation by epithelial cells to cytotoxic T lymphocytes by decreasing transcription levels of SLA Ia mediated antigenic pathway genes. Other genes involved in the immune response, the apoptosis pathway, nucleic acid metabolism and cytoskeleton also appeared to be regulated during PrV infection. The combined approach will help to decipher host response evasion strategies developed by PrV and to study early cellular modifications.

A first-generation microsatellite-based integrated genetic and cytogenetic map for the European rabbit (Oryctolagus cuniculus) and localization of angora and albino.

Although the European rabbit (Oryctolagus cuniculus) is used both in agronomics and in research, genomic resources for this species are still limited and no microsatellite-based genetic map has been reported. Our aim was to construct a rabbit genetic map with cytogenetically mapped microsatellites so as to build an integrated genetic and cytogenetic map. A reference population of 187 rabbits comprising eight three-generation families with 10-25 offspring per family was produced. One hundred and ninety-four of 305 previously identified microsatellites were included in this study. Of these, 158 were polymorphic with two to seven alleles. The map reported here comprises 111 markers, including 104 INRA microsatellites, five microsatellites from another source and two phenotypic markers (angora and albino). Ninety markers were integrated into 20 linkage groups. The remaining 21 microsatellites mapped to separate linkage groups, 19 with a precise cytogenetic position and two with only a chromosomal assignment. The genetic map spans 2766.6 cM and covers 20 rabbit chromosomes, excluding chromosomes 20, 21 and X. The density of this map is limited, but we used it to verify the location of angora and albino on chromosomes 15q and 1q, respectively, in agreement with previously published data. This first generation genetic/cytogenetic map will help gene identification and quantitative trait loci mapping projects in rabbit.

Expanded comparative mapping between man and rabbit and detection of a new conserved segment between HSA22 and OCU4.

Rabbit, a domestic species exploited both in animal production and medical research has only recently begun to be included in gene mapping projects, in particular by the French National Institute of Agronomics. By 2002, less than 60 genes had been precisely localised on rabbit chromosomes, which led us to start a large-scale project on gene mapping in rabbit with the publication of 133 gene localisations in 2003 (Chantry-Darmon et al., 2003). Here, we report the localisation of 102 new genes resulting in good coverage of the rabbit genome and an eight-fold enrichment of the gene map. In addition, we have detected a new conserved segment between rabbit chromosome 4q15.3 and part of human chromosome 22 and thus improved the comparative map with the human genome.

High levels of nucleotide diversity in the European rabbit (Oryctolagus cuniculus) SRY gene.

We have sequenced 2,388 bp of the European rabbit sex determining region Y (SRY) gene. These data provide a 10-fold increase in the coverage of the Y chromosome in this species, including the entire open reading frame of the SRY, the polyadenylation signal, and two repetitive sequences in the 5' -region. A survey of 2021 bp of this gene in eight domestic breeds and four wild individuals revealed a total of nine single nucleotide polymorphisms and one indel, defining two deeply divergent lineages. The resulting estimation of nucleotide diversity (pi=1.34 x10(-3)) is very high when compared with other species, but no variability was detected among the domestic breeds. This study represents a first step in the characterization of the European rabbit Y chromosome and its variability. These sequences can be used in additional phylogeographical analyses of the European rabbit and other Leporid species, as well as in evolutionary studies of sex determination and the Y chromosome in wild species.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.

133 new gene localizations on the rabbit cytogenetic map.

Rabbit (Oryctolagus cuniculus), besides its interest for medical research and biotechnological applications, has a small agronomic production in southern European countries. However, it is still a "map-poor" species with about 80 genes mapped. Recently, useful tools for research on this species have been developed, such as heterologous human-rabbit chromosome painting data and a rabbit BAC library. In this study, our aim is to enrich the rabbit cytogenetic map using the FISH technique. Towards this, we have used cDNAs (rabbit and non rabbit) present in the public databases to determine intra-exon primers used to screen our three-genome equivalent BAC library, by standard PCR directly on DNA pools, and by hybridization of high-density filters. 133 BAC clones containing the genes of interest were isolated and FISH-mapped to the rabbit chromosomes. We present the localization of new genes on all rabbit chromosomes except OCU20 and OCUY and some preliminary data on the rabbit/human comparative map. In addition, this set of BAC clones quite regularly distributed on the rabbit genome will be useful to isolate microsatellites, in order to construct a first generation genetic map.

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit.

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.

A case of intersexuality in pigs associated with a de novo paracentric inversion 9 (p1.2; p2.2).

In several mammalian species, genetic defects can be responsible for the interruption of and/or the deviation from the sequential steps of normal gonadal differentiation, leading to a sex-reversal syndrome. In pigs, female-to-male sex-reversal conditions are particularly frequent, but their aetiologies remain unclear. Chromosomal abnormalities that co-occur with sex-reversal disorders can be useful in the identification of loci containing responsible or susceptibility genes. This report describes a female-to-male SRY-negative intersex pig with a de novo paracentric inversion of the short arm of one chromosome 9 (p1.2; p2.2). We have fine mapped the proximal chromosomal breakpoint of this rearrangement because it corresponded to a region potentially involved in the pig intersexuality. Fluorescent in situ hybridization (FISH) experiments carried out with Bacterial Artificial Chromosome (BAC) clones located within the critical region defined by genetic linkage analysis and ordered on the porcine RH map allowed us to locate the proximal breakpoint between markers SW2571 and SW539. Further investigations are currently in progress to find new markers inside this interval, in order to determine the BAC in which the break occurred.

Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes.

Direct detection of fluorescent in situ hybridization signals on R-banded chromosomes stained with propidium iodide is a rapid and efficient method for constructing cytogenetic maps for species with R-banded standard karyotypes. In this paper, our aim is to establish an R-banded rabbit karyotype nomenclature that is in total agreement with the 1981 G-banded standard nomenclature. For this purpose, we have produced new GTG- and RBG-banded mid-metaphase karyotypes and an updated version of ideograms of R-banded rabbit chromosomes. In addition, to confirm correlations between G- and R-banded chromosomes, we have defined a set of 23 rabbit BAC clones, each containing a specific gene, one marker gene per rabbit chromosome, and we have localized precisely each BAC clone by FISH on both G- and R-banded chromosomes.