Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans

  • Svetlana A. Romanenko ,

    rosa@mcb.nsc.ru (SAR); fy1@sanger.ac.uk (FY)

    Affiliations Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk, Russia, Novosibirsk State University, Novosibirsk, Russia

  • Polina L. Perelman,

    Affiliations Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk, Russia, Novosibirsk State University, Novosibirsk, Russia

  • Vladimir A. Trifonov,

    Affiliations Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk, Russia, Novosibirsk State University, Novosibirsk, Russia

  • Natalia A. Serdyukova,

    Affiliation Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk, Russia

  • Tangliang Li,

    Affiliation State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, PR China

  • Beiyuan Fu,

    Affiliation Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom

  • Patricia C. M. O’Brien,

    Affiliation Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom

  • Bee L. Ng,

    Affiliation Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom

  • Wenhui Nie,

    Affiliation State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, PR China

  • Thomas Liehr,

    Affiliation Jena University Hospital, Institute of Human Genetics and Anthropology, Jena, Germany

  • Roscoe Stanyon,

    Affiliation Department of Biology, University of Florence, Florence, Italy

  • Alexander S. Graphodatsky,

    Affiliations Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk, Russia, Novosibirsk State University, Novosibirsk, Russia

  • Fengtang Yang

    rosa@mcb.nsc.ru (SAR); fy1@sanger.ac.uk (FY)

    Affiliation Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans

  • Svetlana A. Romanenko, 
  • Polina L. Perelman, 
  • Vladimir A. Trifonov, 
  • Natalia A. Serdyukova, 
  • Tangliang Li, 
  • Beiyuan Fu, 
  • Patricia C. M. O’Brien, 
  • Bee L. Ng, 
  • Wenhui Nie, 
  • Thomas Liehr
PLOS
x

Abstract

The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents.

Introduction

The domesticated guinea pig (Cavia porcellus) is a proverbial animal model traditionally used in biomedical research (e.g. [1,2]). The correct diploid number (2n = 64) of C. porcellus was determined more than 60 years ago [3]. Before banding or differential staining became available variants of several chromosomal pairs of guinea pig were reported in the literature [4,5,6,7]. With the advent of C-banding it became clear that these variants were due to differences in the amount of heterochromatin [8]. Later there were a series of more detailed reports on differential staining chromosomes of the chromosomes of C. porcellus [9,10,11,12,13,14,15,16]. However, in spite of the guinea pig's importance in research there is no karyotype standard or chromosome nomenclature for this species.

The lack of genomic resources for the guinea pig compared to other rodents such as the laboratory mouse and rat explains why, over the last decade, the guinea pig has fallen out of favor as a model organism. In contrast, the laboratory rat and mouse were among the first organisms for which high coverage genome assemblies were available.

A low coverage (7x) of the guinea pig genome assembly only became available in 2008 as one of the 29 mammals whose genomes were sequenced by the Mammalian Genome Project [17,18]. Additional sequencing of several guinea pig strains is under way for SNP discovery [18,19]. However, the guinea pig sequence scaffolds have not been anchored to chromosomes in the current assembly, largely due to the lack of large-insert clone-based physical maps (although BACs are available for the guinea pig [20]) and maps of conserved synteny. A well-characterized karyotype and map of conserved synteny with human is the first step towards linking sequencing data and chromosomes.

Comparative cytogenetic maps enable homology links between genomes and transfer of gene mapping information from the well-studied genomes to uncharted genomes of other organisms [21]. Comprehensive chromosome painting data is available to comparatively link the human and representatives of all major mammalian clades (cf. [22,23]). Although comparisons between human and rodent genomes using chromosome painting have had limited success, results in Sciuromorpha, Castorimorpha, and Anomaluromorpha showed that most of the ancestral eutherian syntenic associations were conserved [24,25,26,27,28,29,30,31]. However, some rodents such as Myomorpha have experienced massive chromosomal rearrangements [32]. Currently there is no information about the rate of genomic changes in Hystricomorpha, the taxa to which C. porcellus, belongs. It is thus pertinent and timely to extend chromosome painting to a Hystricomorpha rodent and in particular the guinea pig.

Chromosome painting maps are most often unidirectional. However, cross-species reciprocal painting is more precise and can generate detailed subchromosomal-level comparative maps. In reciprocal painting probes are produced from both species and painting is bi-directional. Currently, painting probe sets are available for only 19 rodent species and, there are only a few reports of reciprocal painting studies in rodents [33,34,35,36,37,38,39]. In spite of the fact that the Hystricomorpha comprises over 260 species up to now only two paint sets were produced (Octodon degus [40] and Heterocephalus glaber [41]). Further, there is still no consensus for the phylogenetic relationship between the guinea pig and other hystricomorphs [42,43,44]. The generation of a set of paint probes for the guinea pig will help resolve phylogenetic relationships within Hystricomorpha, especially when integrated with other biomolecular results [42,44,45,46].

Here, painting probes for domesticated guinea pig were made from two fibroblast cell lines by flow sorting and microdissection. The application of the probes to cross-species reciprocal chromosome painting with human allowed us to establish a comparative chromosome map between domesticated guinea pig and human.

Materials and Methods

Ethics statement

Two primary fibroblast cell lines of C. porcellus (male) were used in this study: the first is an established cell line (Cat number: KBC 200301) that was obtained directly from Kunming Cell Bank (KCB), the Chinese Academy of Sciences, while the second was derived from skin biopsy from a guinea pig obtained from the animal facility at the National Cancer Institute (NCI), USA. For convenience, the former cell line was named as CPO-KCB, while the latter as CPO-NCI.

Tissue sample for CPO-NCI was obtained in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animals were not sacrificed. Instead a small piece of ear, about 1–2 mm square was cut with scissors from the tip of the ear as described in [47]. It was not necessary to use anesthesia or analgesics as very minimal damage or stress was done to the animals and they were immediately released with no ill effects.

Chromosome preparation and chromosome staining

Human metaphases were prepared from a short-term culture of human peripheral lymphocytes stimulated with a combination of three mitogens: pokeweed (Sigma-Aldrich, final concentration: 1%), phytohemagglutinin (Sigma-Aldrich, final concentration: 1%) and conconavalin A (Sigma-Aldrich, final concentration: 1%). The cultures were arrested with colchicine (final concentration: 0.15 μg/ml) for 45 min and chromosomes were harvested using a standard procedure [9].

The C. porcellus cells were cultivated and chromosomal suspensions were made as described previously [47,48]. Metaphase preparations were made as described earlier [49,50]. The GTG- and CBG-bandings were performed as described by [51] and [52], respectively.

Generation of painting probes for C. porcellus

The set of human chromosome specific painting probes has been generated in the Cambridge Resource Centre for Comparative Genomics (UK) and provided for collaborative research use [53].

Painting probes for C. porcellus were generated independently from the two cell lines mainly by flow sorting. The flow sorting of CPO-KCB was done using a FACStar Plus (Becton Dickinson) at the University of Cambridge [54] as well as using a MoFlo Cell Sorter (Beckman Coulter) at the Wellcome Trust Sanger Institute (UK) [55]. Painting probes from both CPO-KCB were made using the conventional 6-MW primer (5’-CCG ACT CGA GNN NNN NAT GTG G-3’) and were labeled with biotin- and digoxigenin-dUTP (Roche) as well or directly with Cy3-, Cy5- or Green-dUTPs (Jena BioScience) by DOP-PCR [54,56]. The chromosomes of CPO-NCI were sorted using FACS Vantage SE (Becton Dickinson) at the National Cancer Institute (USA) [57]. Four different DOP-primers (6MW, FS (5′-CGG ACT CGA GNN NNN NTA CAC C-3′), GAG (5′-GAG GAG GAG GAG GAG GAG GAG -3′), G1/G2 (5′-GAG GAT GAG GTT GAG NNN NNN TGG-3′/5′-GTG AGT GAG AGG ATG AGG TTG AG-3′)) were used for chromosome test sorts. As G1/G2 primers produced the best paints with a minimum of cross hybridization they were selected to amplify the entire set of flow-sorted chromosomes. First round of PCR was made with primer G1 and the second round of PCR with primer G2 [57].

Microdissection was used to generate chromosome-specific probes for such CPO-NCI chromosomes that could not be resolved into single-chromosome-containing peaks by flow cytometry at the Institute of Human Genetics and Anthropology (Germany) as previously described [58]. All microdissected probes except CPO Xp and Xq were generated using GenomePlex Whole Genome Amplification (WGA1) Kit (Sigma-Aldrich) [59]. For CPO Xp and Xq generation DOP-PCR was used.

Telomeric and ribosomal DNA probes

The telomeric DNA probe was generated by PCR using the oligonucleotides (TTAGGG)5 and (CCCTAA)5 [60]. Clones of human ribosomal DNA containing the complete 18S-rRNA and 28S-rRNA genes were obtained as described [61].

Fluorescence in situ hybridization (FISH)

We used sequential GTG-banding [51] and FISH or simultaneous DAPI-banding and FISH for precise chromosome identification [49,50]. Four digital imaging systems were used in this study: VideoTesT-FISH and VideoTesT-Karyo (VideoTesT, Saint-Petersburg, Russia), Case Data Manager (Applied Spectral Imaging Inc.), CytoVision system (Applied Imaging Corp.), and SmartCapture and SmartType Karyotyper (Digital Scientific, UK). Hybridization signals were assigned to specific chromosome regions defined by GTG- or DAPI-banding patterns.

Results

Generation of chromosome-specific painting probes and assignment of probes onto G-banded chromosomes

The karyotype of C. porcellus contained many chromosomes of similar sizes and morphology (Fig 1), which represented a technical challenge for chromosome sorting by flow cytometry. Chromosome suspensions from the two cell lines of C. porcellus (CPO-KCB and CPO-NCI) were independently subjected to flow-sorting (Fig 2A–2C), which returned two sets of probes, each with its own characteristics. The chromosomes of CPO-KCB were resolved into 28 peaks (Fig 2A and 2B). The set of probes from CPO-KCB generated by DOP-PCR with the 6MW primer often gave strong cross-hybridization signals onto the short arms of the biarmed autosomes as well as to the largely C-band positive regions on chromosomes 21, Xp-proximal, and Yq, as demonstrated by the hybridization patters of CPO10 (Fig 2D). These probes were identified mainly by sequential G-banding and multi-colour FISH. The quality of this set of probes varied when applied to cross-species painting; for probes that did not work well when painted onto human chromosomes, new probes were made using a new batch of flow-sorted chromosomes. Although CPO-KCB was sorted using two different cell sorters (i.e. FACStar Plus vs. MoFlo), no obvious difference in resolution was found in the flow karyotypes.

thumbnail
Fig 1. Idiogram and karyotype of the guinea pig with homologies to human (HSA) revealed by comparative chromosome painting.

Black dots mark position of centromeres. Black triangles indicate NORs located on CPO1, CPO6 and CPO11 (see comments in the text). Each pair presented in two copies to show chromosomes with different resolution: KCB—CPO-KCB, NCI—KPO-NCI.

https://doi.org/10.1371/journal.pone.0127937.g001

thumbnail
Fig 2. Bivariate flow karyotypes of C. porcellus and characteristic hybridization patterns of paint probes.

a and b—Flow karyotype of CPO-KCB cell line, a shows the annotated top chromosomes and b show an enlarged version of the inferior part of the flow karyotype (in grey frame), c—CPO-NCI cell line, d—hybridization pattern of CPO10 paint generated using 6MW primer, e— CPO2 (green) and CPO14+18 (red) paints (indicated by arrows) from CPO-NCI cell line generated with G1 and G2 primers on metaphase chromosomes from C. porcellus. Arrows indicate the painting results in Fig 2d and 2e.

https://doi.org/10.1371/journal.pone.0127937.g002

Two separate attempts were made to sort the chromosomes of CPO-NCI, resolving the chromosomes into 26 peaks (Fig 2C) and 28 peaks, respectively. Applying a tighter sorting gate on the flow chart only led to a slight reduction in the number of peaks containing multi-chromosomes. The content of each peak was determined by hybridizing the paint probes back onto G-banded chromosomes of the C. porcellus. Both sets of probes made using the G1 and G2 primer did not produce cross hybridization signals to the C-band positive heterochromatic blocks and the repeat-rich short arms of the biarmed autosomes (Fig 2E). Indeed, at NCI, Frederick, USA, chromosome test sorts were first amplified by a number of different DOP-primers (6MW, FS, GAG, G1/G2). Then the primer that produced the best paints with a minimum of cross hybridization was selected to amplify the entire set of flow-sorted chromosomes. In this case the cleanest results were produced by DOP-PCR with G1 and G2 primers which enabled the avoidance of excessive amplification of heterochromatin. However, chromosomes 12 and Y were apparently “missing” from the flow karyotype of CPO-NCI, perhaps due the use of over-tight sorting gates.

The GTG-banded karyotype of C. porcellus has been reported before [9,10,12,14,15]. Nevertheless, apart from a few large-sized chromosomes and X, the majority of the medium- and small-sized chromosomes were almost impossible to identify unambiguously by banding patterns alone. Furthermore, as demonstrated by flow cytometry (Fig 2A–2C), the same chromosomes, due to the variation in heterochromatin, often appeared in different positions in the two flow karyotypes. Notably, the positions of most medium-sized chromosomes (CPO 4–24) in the two flow karyotypes (Fig 2A–2C) often appeared to be “inconsistent” when they were compared across the two cell lines. Nevertheless, such an apparent inconsistency was due to variations in the heterochromatic short arms among individuals and as such was true reflection of chromosome short arm heteromorphisms between individuals [8]. It was difficult to estabish the correspondence between all the chromosomes of CPO-KCB and CPO-NCI on the basis of G-banding and painting (Fig 1). Indeed, it was possible to unambiguously establish the G-banded chromosome correspondence between some chromosomes only after hybridizing both sets of painting probes onto human metaphases. Here we opted to establish a new chromosome nomenclature, because it was impossible to follow published karyotypes with confidence. We arranged chromosomes mainly according to size.

Since the majority of the C. porcellus chromosomes could not be unequivocally identified by GTG- or DAPI-banding alone and many autosomes could not be sorted separately, we also made a set of probes derived from microdissected CPO-NCI chromosomes, in order to increase the resolution of C. porcellus probes. We generated six subchromosome specific probes for the three largest CPO chromosomes—CPO1, 2 and X: for chromosomes CPO1 and CPO2 we obtained probes for proximal and distal parts of the q-arms; while for the X chromosome we prepared probes for p- and q-arms. The p-arm of the X chromosome contains a huge heterochromatic block, giving a strong hybridization signal on CPOY heterochromatin. Chromosome-specific probes were also made by microdissection of 16 autosomes (CPO5, 6, 7, 9, 10, 11, 13, 14, 17, 20, 21, 22, 23, 27, 29 and 30). The quality of probes varied, with some highlighting the heterochromatic regions on other chromosomes, but they were useful for reliable identification of co-sorted CPO chromosomes.

Standardization of C. porcellus karyotype

The precise comparison of GTG-banded chromosomes obtained in different laboratories was the first step in the construction of the comparative chromosome map for human and guinea pig. Summarizing the guinea pig karyotype description based on published data and presented here we can conclude that: the diploid chromosome number is 2n = 64 and the fundamental number of chromosomal arms is FN = 92. The X is a medium size submetacentric and the Y is a small size acrocentric with a large block of heterochromatin on the q-arm. Some chromosome pairs are NOR-bearing (see below) and several chromosomal pairs possess large blocks of heterochromatin with a variation in size even between homologues. Telomeric repeats are concentrated in pericentromeric regions. To solve the problem of chromosome correspondence in different reports in the literature we introduce here a nomenclature of C. porcellus GTG-banded chromosomes. The nomenclature is made in reference to an ideogram of C. porcellus chromosomes and in reference to human chromosome homology (Fig 1, S1 Fig). All pairs of autosomes were placed in order of decreasing size. Additionally, development of a set of molecular markers (for example, BAC markers) for easy molecular identification of each chromosome through FISH would be beneficial for Cavia cytogenetics. For now the combined use of Cavia chromosome-specific probes developed here and human chromosome probes has enabled the unambiguous identification of each of the guinea pig chromosomes.

CBG-banding in CPO-NCI

We observed the same CBG-banding pattern for C. porcellus chromosomes, as that reported previously [9,13,14]. CBG-banding revealed that all autosomes in C. porcellus karyotype had rather large C-positive pericentromeric blocks. A pair of small-sized autosomes (CPO21) had large blocks of pericentromeric heterochromatin on both arms. The C. porcellus Y-chromosome was C-positive and X-chromosomes had pericentromeric blocks on the p-arms (Fig 3).

thumbnail
Fig 3. CBG-banding of C. porcellus metaphase chromosomes.

Figures were constructed according to chromosome size and CBG-banding.

https://doi.org/10.1371/journal.pone.0127937.g003

Distribution pattern of telomeric repeats and ribosomal DNA in CPO-NCI

Clusters of telemetric sequences were found not only at telomeres of chromosomes but were also present at the large pericentromeric regions and heterochromatic blocks in addition to the main pattern of localization of telomeric repeats as reported by Meyne et al. ([62]). The use of early metaphases with longer chromosomes allowed the detection of some more detailed features, revealing one to three clusters on each chromosome at the pericentromeric region. We localized the 18S-rRNA/28S-rRNA probe by itself and in dual-color FISH with a telomeric probe (Fig 4A). We observed unusual intermittent patterns of telomeric and NOR signals on several chromosomes. For example, a NOR co-localized with a large telomeric block on the distal part of the p-arm on CPO1. But it apparently shows some heteromorphism between two homologues of one pair: the NOR signal is distinctly seen on one homologue and is covered by the telomeric signal on the other. Possibly it explains the heteromorphism of CPO1 p-arm described in earlier works on the C. porcellus karyotype [7,63]. Our NOR localization through FISH following GTG-banding and co-localization with selected chromosome-specific probes allowed us to determine that chromosomes 1 and 11 carry NOR on the short arm. While most of the NORs are located on the distal part of p-arms, one chromosome (CPO6) appears to have a NOR on both p- and q-arms (Fig 4A). In all we observed 7–8 pares of NOR-bearing chromosomes. All but one NOR-bearing pairs of chromosomes carry large clusters of telomeric sequences. Additional studies are required to determine the precise localization of other NOR sites.

thumbnail
Fig 4. Examples of fluorescent in situ hybridization.

a—localization of telomeric DNA probe (red) and rDNA probe (green) on metaphase chromosomes from C. porcellus; telomeric signals are too weak to capture without risking over-exposure to the strong signals from pericentromeric regions; b—HSA19 (green) and CPO6+7 (red) on metaphase chromosome from C. porcellus; c—CPO26 (green) and CPO19 (red) on metaphase chromosome from H. sapiens; d—HSA22 (green) and CPO14+16 (red) on metaphase chromosomes from C. porcellus; e—HSA20 (green) and CPO6+7 (red) on metaphase chromosomes chromosome from C. porcellus; f—CPO14 on metaphase chromosomes from H. sapiens. Arrows indicate the corresponding human chromosomes in 4b, 4d, 4e and the corresponding C. porcellus chromosomes in 4c and 4f. Scale bars indicate 10 μm.

https://doi.org/10.1371/journal.pone.0127937.g004

Reciprocal chromosome painting between human and C. porcellus

Reciprocal painting between human and C. porcellus generated high-resolution comparative chromosome maps. The hybridization of 22 human autosomal probes onto the C. porcellus metaphase chromosomes revealed 78 homologous segments in the C. porcellus genome (Fig 1). The following associations of synteny-conserved human chromosome segments were detected in the guinea pig genome: HSA 1/8, 1/14/2, 2/4/2/4, 2/9/1/9/15/14, 3/4, 3/8, 3/10/6, 3/11/19, 3/20/3, 3/21/3, 4/13/16/4/16, 5/8 (twice), 5/10/7, 5/13, 6/1/19, 6/10, 8/4/8, 11/17/7/1/15, 12/15, 12/22, 12/22/16, 17/10/17/10/17 (Fig 1). The “reverse” painting from C. porcellus autosomal probes (see examples of fluorescence in situ hybridization in Fig 4B–4F) also detected 78 conserved synteny segments in the human genome (Fig 5). Paints made from both C. porcellus lines were hybridized onto human chromosomes, which also served as a common reference point for double checking the correspondence of flow peaks in the flow karyotypes of CPO-NCI and CPO-KCB. As expected, the heterochromatic regions on both human and C. porcellus chromosomes were not hybridized by any chromosomal probes in reciprocal painting between distantly related species (or cross-order reciprocal painting).

thumbnail
Fig 5. Human karyotype with homologies to guinea pig (CPO) revealed by painting.

Black dots mark positions of centromeres.

https://doi.org/10.1371/journal.pone.0127937.g005

Discussion

The guinea pig, Cavia porcellus, was one of the most important biomedical animal models in the last century. It lost popularity in part due to a lack of modern genomic tools to fully exploit this animal model. In contrast, the mouse was the first mammalian species after human (2001) for which, only one year later (2002), a complete genome assembly was available. The genome of guinea pig was only sequenced in 2008, but without even assigning scaffolds to actual chromosomes. There was no data to relate the karyotype of the guinea pig to human before this report.

Here we generated sets of chromosome painting probes for the guinea pig (C. porcellus) and established homology maps between this species, human and by extension other rodents. The great phylogenetic distance between hystricomorph rodents and primates (the divergence time between the ancestor of rodents and the common ancestor of primates and artiodactyls is about 80–100 Myr [64,65]), together with extensive rearrangements between humans and C. porcellus, made the FISH-comparison difficult. Due to their similar size multiple chromosomes were found in many peaks of the flow karyotype. Chromosomes were difficult to cytogenetically identify due to similar size and shape, a lack of distinctive banding patterns, and in some case, hybridization quality. Only the combination of expertise and resources of different laboratories enabled us to overcome these difficulties and achieve a full and precise comparison between the karyotypes of guinea pig and human.

Ancestral associations found in the guinea pig

Among associations of synteny-conserved human chromosome segments detected in the guinea pig genome by reciprocal chromosome painting (Fig 1) only five of them are common for the guinea pig and the putative eutherian/rodent ancestral karyotypes [25]: HSA 3/21, 8/4/8, 12/22 (twice), and 14/15. It is very likely that these syntenies are homologous in the guinea pig and other rodents; however, only future research will verify if these breakpoints and syntenies have identical origins. In contrast, the HSA7/16 and 16/19 associations presumed ancestral for eutherians were not found. Furthermore, three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were also not found in C. porcellus. The most likely hypotheses to explain these findings are that 1) the associations were lost in the evolutionary line leading to the guinea pig due to a high rate of chromosome evolution, or 2) the size of the associations in the guinea pig were below the resolution of techniques used in our study.

Level of karyotype divergence in the guinea pig

The number of autosomal conserved segments between two species can serve as a measure of karyotype divergence. Rodents branched out of the mammalian tree about 100 MYA according to molecular clock estimates, and after another 40 MYA Hystricomorpha have diverged from other rodent suborders [66]. In the order Rodentia the number of conservative segments revealed by human painting probes varies from 36–37 fragments in squirrels to 95 in mouse and rat genomes [32]. This range of differences results from at least two modes of genome reorganization in rodents: a slow, conservative evolution in Sciuromorpha and a high evolutionary rate in Myomorpha which disrupted chromosomal syntenies.

Our reciprocal chromosome painting and GTG-banding comparisons between guinea pig and human allowed us to estimate the level of chromosomal divergence of the guinea pig karyotype. Using human painting probe we found a total of 78 segments of homology between guinea pig and human. We can conclude that the guinea pig has a high rate of chromosome evolution. This rate is comparable to but somewhat lower than that found in myomorphs (78 vs 95 segments found in mouse).

Future use of guinea pig painting probes in phylogenomics

The painting probe sets developed from the guinea pig will also allow more incisive studies of hystricomorph chromosome evolution and allow comparison between hystricomorphs and other rodent taxa. There are very few studies that describe karyotypes of the closest relatives of guinea pig [67,68,69]. In general all members of the genus Cavia have a diploid number 2n = 64, except the island species C. intermedia with 2n = 62 [67]. Apparently there is a great deal of variation in the amount and localization of heterochromatic blocks between different species. To our knowledge, there are no studies that directly compared differentially stained chromosomes between Caviidae species. There are no studies comparing the guinea pig chromosomes with those of other mammals. Compared with all other rodent groups, Hystricomorpha, which include among others the porcupines, chinchillas, pacas, agoutis and capybaras, is far less well-studied by modern molecular cytogenetic approaches [40,41,67,70].

We hypothesize that hystricomorph rodents in general are probably characterized by much higher rates of genome reorganization than most mammals. Moreover, as some of the ancestral syntenies may have been disrupted in the karyotype of C. porcellus, it is possible that they were also disrupted in the phylogenetic branch of Caviidae or, may be, in all hystricomorphs. To confirm or reject this hypothesis it is necessary to have a taxonomically rich array of hystricomorph species from different families.

Future use of guinea pig probes in biomedical research

High-resolution synteny maps between human and laboratory mouse and rat, based on genome-wide sequence comparison, are available from ENSEMBL. The comparative chromosome map presented here should allow the inference of genome-wide chromosomal correspondence between guinea pig and laboratory mouse and rat, and many other species using human chromosome as the common reference, a great potential awaiting to be fully explored. Indeed, the breeding of the guinea pig as a pet is on the rise with over 20 breeds and many varieties involving coat color and hair properties being currently established, opening room to study pigmentation and hair growth related genes, their mutations and related disorders [71]. The economic importance of the guinea pig should not be ignored. They are an important meat source in South America that may represent interest for genetic characterization of meat-related genes.

Conclusions

Here we present the first report on chromosome painting between human and hystricomorph rodents. Further comprehensive GTG-banding and chromosome painting studies between different representative species of this group would shed light on hystricomorph karyotype evolution and will help to establish phylogenetic relationships both within and between rodent suborders. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig and will facilitate the use of the guinea pig as a model for human diseases.

Supporting Information

S1 Fig. The G-banded ideogram complete with band nomenclature.

p and q—short and long arms of chromosome, respectively. Bigger figures mark segments, smaller—separate bands.

https://doi.org/10.1371/journal.pone.0127937.s001

(TIF)

Acknowledgments

One set of Guinea pig probes was developed when RS and PLP were at the National Cancer Center-Frederick with the collaboration of Gary Stone (now unfortunately defunct). The authors would like to thank the reviewers for comments helpful in improving this report.

Author Contributions

Conceived and designed the experiments: ASG FY. Performed the experiments: BF BLN FY NAS PCMOB PLP RS SAR T. Liehr T. Li VAT WN. Analyzed the data: SAR FY. Contributed reagents/materials/analysis tools: BF PLP SAR. Wrote the paper: ASG SAR FY RS. Proofread the paper: PCMOB PLP T. Li VAT WN.

References

  1. 1. Ohi Y, Kimura S, Haji A. Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius. Brain Res. 2014; 1581: 12–22. pmid:24792310
  2. 2. Saidullah B, Muralidhar K, Fahim M. Onset of diabetes modulates the airway smooth muscle reactivity of guinea pigs: role of epithelial mediators. J Smooth Muscle Res. 2014; 50: 29–38. pmid:24829035
  3. 3. Makino S. Notes on the chromosomes of four species of small mammals. J Fac Sci Hokkaido Univ. 1947; 9: 345–357.
  4. 4. Awa A, Sasaki M, Takayama S. An in vitro study of the somatic chromosomes in several mammals. Jap J Zool. 1959; 12: 257.
  5. 5. Hsu TC, Pomerat CM. Mammalian chromosomes in vitro: II. A method for spreading the chromosomes of cells in tissue culture. Journal of Heredity. 1953; 44: 23–30.
  6. 6. Manna GK, Talukdar M. Chromosomal polymorphism in the guinea-pig, Cavia porcellus. Experientia. 1964; 20: 324–325. pmid:5855858
  7. 7. Ohno S, Weiler C, Stenius C. A dormant nucleolus organizer in the guinea pig, Cavia cobaya. Exp Cell Res. 1961; 25: 498–503. pmid:14481404
  8. 8. Blanchi NO, Ayres J. Polymorphic patterns of heterochromatin distribution in guinea pig chromosomes. Chromosoma. 1971; 34: 254–260. pmid:5112132
  9. 9. Graphodatsky AS, Radjabli SI. Chromosomes of agricultural and laboratory mammals. Novosibirsk: Nauka; 1988.
  10. 10. Graphodatsky S (2006) Cavia porcellus (cobaya). In: O’Brien SJ, editor. Atlas of mammalian chromosomes: A John Wiley & Sons, Inc. pp. 324.
  11. 11. Lomholt B, Christensen K, Frederiksen S. Guinea pig (Cavio cambayo) 5S rRNA genes map to 7q2, 20q2 and 30q2 shown by an R-banded karyotype with PNA-FISH. Hereditas. 2002; 136: 104–107. pmid:12369094
  12. 12. Moore DE, Evans WH, Mage MG. A deletion in chromosome 1 in cells of a transplantable granulocytic leukemia (GL-13) in guinea pigs. J Natl Cancer Inst. 1982; 69: 591–594. pmid:6955556
  13. 13. Natarajan AT, Raposa T. Repetitive DNA and constitutive heterochromatin in the chromosomes of guinea pig. Hereditas. 1974; 76: 145–147. pmid:4136007
  14. 14. Popescu NC, Evans CH, DiPaolo JA. Chromosome patterns (G and C bands) of in vitro chemical carcinogen-transformed guinea pig cells. Cancer Res. 1976; 36: 1404–1413. pmid:1260765
  15. 15. Whang-Peng J, Lee EC, Forni G, Green I. Mutant lines of guinea pig L2C leukemia. II. Comparative cytogenetic studies and banding analyses of normal and leukemic karyotypes. J Natl Cancer Inst. 1976; 57: 897–905. pmid:1034020
  16. 16. Zenzes MT, Schmid M, Engel W. Silver-stained nucleolus organizers in the guinea pig, Cavia cobaya. Cytogenet Cell Genet. 1977; 19: 368–372. pmid:76529
  17. 17. Broad Institute. 29 Mammals Project. Available: http://www.broadinstitute.org/scientific-community/science/projects/mammals-models/29-mammals-project.
  18. 18. Broad Institute. Guinea Pig Genome Project. Available: http://www.broadinstitute.org/science/projects/mammals-models/guinea-pig/guinea-pig.
  19. 19. Genome Bioinformatics Group. Guinea pig (Cavia porcellus) Genome Browser. 2008. Available: http://genome.cse.ucsc.edu/cgi-bin/hgGateway?org=Guinea+pig&db=cavPor3&hgsid=172184341.
  20. 20. BACPAC Resources Center. pp. CHORI-234: Guinea pig (F) (Cavia porcellus) BAC Library.
  21. 21. Chowdhary BP, Raudsepp T, Fronicke L, Scherthan H. Emerging patterns of comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Research. 1998; 8: 577–589. pmid:9647633
  22. 22. Ferguson-Smith MA, Trifonov V. Mammalian karyotype evolution. Nat Rev Genet. 2007; 8: 950–962. pmid:18007651
  23. 23. Graphodatsky A, Ferguson-Smith MA, Stanyon R. A short introduction to cytogenetic studies in mammals with reference to the present volume. Cytogenet Genome Res. 2012; 137: 83–96. pmid:22846392
  24. 24. Beklemisheva VR, Romanenko SA, Biltueva LS, Trifonov VA, Vorobieva NV, Serdukova NA, et al. Reconstruction of karyotype evolution in core Glires. I. The genome homology revealed by comparative chromosome painting. Chromosome Res. 2011; 19: 549–565. pmid:21559983
  25. 25. Graphodatsky AS, Yang F, Dobigny G, Romanenko SA, Biltueva LS, Perelman PL, et al. Tracking genome organization in rodents by Zoo-FISH. Chromosome Res. 2008; 16: 261–274. pmid:18266061
  26. 26. Li T, O'Brien PC, Biltueva L, Fu B, Wang J, Nie W, et al. Evolution of genome organizations of squirrels (Sciuridae) revealed by cross-species chromosome painting. Chromosome Res. 2004; 12: 317–335. pmid:15241012
  27. 27. Li T, Wang J, Su W, Nie W, Yang F. Karyotypic evolution of the family Sciuridae: inferences from the genome organizations of ground squirrels. Cytogenet Genome Res. 2006; 112: 270–276. pmid:16484783
  28. 28. Richard F, Dutrillaux B. Low, complex and probably reticulated chromosome evolution of Sciuromorpha (Rodentia) and Lagomorpha. Cytogenet Genome Res. 2012; 137: 218–232. pmid:22846378
  29. 29. Richard F, Messaoudi C, Bonnet-Garnier A, Lombard M, Dutrillaux B. Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes. Chromosome Res. 2003; 11: 597–603. pmid:14516068
  30. 30. Sannier J, Gerbault-Seureau M, Dutrillaux B, Richard FA. Conserved although Very Different Karyotypes in Gliridae and Sciuridae and Their Contribution to Chromosomal Signatures in Glires. Cytogenetic and Genome Research. 2011; 134: 51–63. pmid:21430366
  31. 31. Stanyon R, Stone G, Garcia M, Froenicke L. Reciprocal chromosome painting shows that squirrels, unlike murid rodents, have a highly conserved genome organization. Genomics. 2003; 82: 245–249. pmid:12837274
  32. 32. Romanenko SA, Perelman PL, Trifonov VA, Graphodatsky AS. Chromosomal evolution in Rodentia. Heredity (Edinb). 2012; 108: 4–16.
  33. 33. Badenhorst D, Dobigny G, Adega F, Chaves R, O'Brien PC, Ferguson-Smith MA, et al. Chromosomal evolution in Rattini (Muridae, Rodentia). Chromosome Res. 2011; 19: 709–727. pmid:21850459
  34. 34. Mlynarski EE, Obergfell CJ, Rens W, O'Brien PC, Ramsdell CM, Dewey MJ, et al. Peromyscus maniculatus-Mus musculus chromosome homology map derived from reciprocal cross species chromosome painting. Cytogenet Genome Res. 2008; 121: 288–292. pmid:18758174
  35. 35. Romanenko SA, Perelman PL, Serdukova NA, Trifonov VA, Biltueva LS, Wang J, et al. Reciprocal chromosome painting between three laboratory rodent species. Mamm Genome. 2006; 17: 1183–1192. pmid:17143584
  36. 36. Stanyon R, Yang F, Cavagna P, O'Brien PC, Bagga M, Ferguson-Smith MA, et al. Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats. Cytogenet Cell Genet. 1999; 84: 150–155. pmid:10393417
  37. 37. Ventura K, O'Brien PC, Yonenaga-Yassuda Y, Ferguson-Smith MA. Chromosome homologies of the highly rearranged karyotypes of four Akodon species (Rodentia, Cricetidae) resolved by reciprocal chromosome painting: the evolution of the lowest diploid number in rodents. Chromosome Res. 2009; 17: 1063–1078. pmid:19936950
  38. 38. Veyrunes F, Dobigny G, Yang F, O'Brien PC, Catalan J, Robinson TJ, et al. Phylogenomics of the genus Mus (Rodentia; Muridae): extensive genome repatterning is not restricted to the house mouse. Proc Biol Sci. 2006; 273: 2925–2934. pmid:17015352
  39. 39. Yang F, O'Brien PC, Ferguson-Smith MA. Comparative chromosome map of the laboratory mouse and Chinese hamster defined by reciprocal chromosome painting. Chromosome Res. 2000; 8: 219–227. pmid:10841049
  40. 40. Svartman M, Stone G, Stanyon R. Molecular cytogenetics discards polyploidy in mammals. Genomics. 2005; 85: 425–430. pmid:15780745
  41. 41. Deuve JL, Bennett NC, Britton-Davidian J, Robinson TJ. Chromosomal phylogeny and evolution of the African mole-rats (Bathyergidae). Chromosome Res. 2008; 16: 57–74. pmid:18293105
  42. 42. Cao Y, Adachi J, Yano TA, Hasegawa M. Phylogenetic place of Guinea-Pigs—no support of the rodent-polyphyly hypothesis from maximum-likelihood analyses of multiple protein sequences. Molecular Biology and Evolution. 1994; 11: 593–604. pmid:8078399
  43. 43. Honeycutt RL, Rowe DL, Gallardo MH. Molecular systematics of the South American caviomorph rodents: relationships among species and genera in the family Octodontidae. Molecular Phylogenetics and Evolution. 2003; 26: 476–489. pmid:12644405
  44. 44. Huchon D, Douzery EJP. From the old world to the new world: A molecular chronicle of the phylogeny and biogeography of Hystricognath rodents. Molecular Phylogenetics and Evolution. 2001; 20: 238–251. pmid:11476632
  45. 45. Dunnum JL, Salazar-Bravo J. Molecular systematics, taxonomy and biogeography of the genus Cavia (Rodentia: Caviidae). Journal of Zoological Systematics and Evolutionary Research. 2010; 48: 376–388.
  46. 46. Spotorno AE, Valladares JP, Marin JC, Zeballos H. Molecular diversity among domestic guinea-pigs (Cavia porcellus) and their close phylogenetic relationship with the Andean wild species Cavia tschudii. Revista Chilena De Historia Natural. 2004; 77: 243–250.
  47. 47. Stanyon R, Galleni L. A rapid fibroblast-culture technique for high-resolution karyotypes. Bollettino Di Zoologia. 1991; 58: 81–83.
  48. 48. Sitnikova NA, Romanenko SA, O'Brien PC, Perelman PL, Fu B, Rubtsova NV, et al. Chromosomal evolution of Arvicolinae (Cricetidae, Rodentia). I. The genome homology of tundra vole, field vole, mouse and golden hamster revealed by comparative chromosome painting. Chromosome Res. 2007; 15: 447–456. pmid:17497247
  49. 49. Graphodatsky AS, Sablina OV, Meyer MN, Malikov VG, Isakova EA, Trifonov VA, et al. Comparative cytogenetics of hamsters of the genus Calomyscus. Cytogenet Cell Genet. 2000; 88: 296–304. pmid:10828614
  50. 50. Yang F, O'Brien PC, Milne BS, Graphodatsky AS, Solanky N, Trifonov V, et al. A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics. 1999; 62: 189–202. pmid:10610712
  51. 51. Seabright M. A rapid banding technique for human chromosomes. Lancet. 1971; 11: 971–972.
  52. 52. Sumner A. A rapid technique for demonstrating centromeric heterochromatin. Exp Cell Res. 1972; 75: 304–306. pmid:4117921
  53. 53. Ferguson-Smith MA. Genetic analysis by chromosome sorting and painting: phylogenetic and diagnostic applications. Eur J Hum Genet. 1997; 5: 253–265. pmid:9412781
  54. 54. Yang F, Carter NP, Shi L, Ferguson-Smith MA. A comparative study of karyotypes of muntjacs by chromosome painting. Chromosoma. 1995; 103: 642–652. pmid:7587587
  55. 55. Nie WH, O'Brien PCM, Ng BL, Fu BY, Volobouev V, Carter NP, et al. Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes)—An atypical species with low diploid number. Chromosome Research. 2009; 17: 99–113. pmid:19172404
  56. 56. Telenius H, Tunnacliffe A, Pelmear AH, Carter NP, Fergusonsmith MA, Nordenskjold M, et al. General amplification of DNA by degenerate jligonucleotide-primed polymerase chain-reaction (DOP-PCR)—a method for mapping and cloning. Cytogenetics and Cell Genetics. 1991; 58: 2154–2154.
  57. 57. Fronicke L, Wienberg J, Stone G, Adams L, Stanyon R. Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc Biol Sci. 2003; 270: 1331–1340. pmid:12965023
  58. 58. Weimer J, Kiechle M, Arnold N. FISH-microdissection (FISH-MD) analysis of complex chromosome rearrangements. Cytogenet Cell Genet. 2000; 88: 114–118. pmid:10773683
  59. 59. Kosyakova N, Hamid AB, Chaveerach A, Pinthong K, Siripiyasing P, Supiwong W, et al. Generation of multicolor banding probes for chromosomes of different species. Molecular Cytogenetics. 2013; 6.
  60. 60. Ijdo JW, Wells RA, Baldini A, Reeders ST. Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res. 1991; 19: 4780. pmid:1891373
  61. 61. Maden BE, Dent CL, Farrell TE, Garde J, McCallum FS, Wakeman JA. Clones of human ribosomal DNA containing the complete 18 S-rRNA and 28 S-rRNA genes. Characterization, a detailed map of the human ribosomal transcription unit and diversity among clones. Biochem J. 1987; 246: 519–527. pmid:3689320
  62. 62. Meyne J, Baker RJ, Hobart HH, Hsu TC, Ryder OA, Ward OG, et al. Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma. 1990; 99: 3–10. pmid:2340757
  63. 63. Schmid W. Heterochromatin in mammals. Arch Julius Klaus Stift Vererbungsforsch Sozialanthropol Rassenhyg. 1967; 42: 1–60. pmid:5632936
  64. 64. Huchon D, Madsen O, Sibbald MJ, Ament K, Stanhope MJ, Catzeflis F, et al. Rodent phylogeny and a timescale for the evolution of Glires: evidence from an extensive taxon sampling using three nuclear genes. Molecular Biology and Evolution. 2002; 19: 1053–1065. pmid:12082125
  65. 65. Li WH, Gouy M, Sharp PM, O'HUigin C, Yang YW. Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora and molecular clocks. Proc Natl Acad Sci U S A. 1990; 87: 6703–6707. pmid:2395871
  66. 66. Adkins RM, Walton AH, Honeycutt RL. Higher-level systematics of rodents and divergence time estimates based on two congruent nuclear genes. Mol Phylogenet Evol. 2003; 26: 409–420. pmid:12644400
  67. 67. Gava A, Freitas TRO, Olimpio J. A new karyotype for the genus Cavia from a southern island of Brazil (Rodentia—Caviidae). Genetics and Molecular Biology. 1998; 21.
  68. 68. George W, Weir BJ, Bedford J. Chromosome studies in some members of the family Caviidae (Mammalia: Rodentia). J Zool Soc Lond. 1972; 168: 81–89.
  69. 69. Maia V. Karyotypes of three species of Caviinae (Rodentia, Caviidae). Exerementia. 1984; 40: 564–566.
  70. 70. Deuve JL, Bennett NC, O'Brien PC, Ferguson-Smith MA, Faulkes CG, Britton-Davidian J, et al. Complex evolution of X and Y autosomal translocations in the giant mole-rat, Cryptomys mechowi (Bathyergidae). Chromosome Res. 2006; 14: 681–691. pmid:16964575
  71. 71. British cavi council. Breed standards for full and guide standard cavies. 2009. Available: http://britishcavycouncil.org.uk/Download/breedStandard.pdf.