Animal Cytogenetics, Gene Mapping and Chromosome Research – The 21st Colloquium Ischia, Naples, Italy

Organizer Leopoldo Iannuzzi and the Animal Cytogenetics Conference Poster
Organizer Leopoldo Iannuzzi and the Animal Cytogenetics Conference Poster

Cytogenetics of animals is moving forward rapidly with the integration of sequencing and robust phylogenies with methods of chromosome analysis. The 21st International Colloquium on Animal Cytogenetics and Gene Mapping held June 2014 covered a range of topics in comparative, molecular, veterinary and environmental cytogenetics, as well as a session on cytogenetics of non-mammalian vertebrates and invertebrates which was much larger than in previous years. Organizer Leopoldo Iannuzzi opened the conference with a discussion of the development of the field seen through the series of colloquiums, now embracing genomics as well as chromosomes and cytogenetics. Abstracts are published by Chromosome Research, and a version of this review is in the Newsletter of the European Cytogeneticists Association, ECA issue 34 July 2014.

The conference also marked the half century since Ingemar Gustavson identified the rob(1;29) translocation in cattle, dramatically opening the field of modern domestic animal cytogenetics, and M Switonski from Poznal, Poland showed development of this research from the first paper in August 1964 to the current status.

Malcolm Ferguson-Smith presenting work on comparative cytogenetics and implications in farm animals
Malcolm Ferguson-Smith presenting work on comparative cytogenetics and implications in farm animals

Malcolm Ferguson-Smith (Cambridge, UK) gave an impressive opening talk, pulling out many key results from his own work of molecular cytogenetics that is relevant to veterinary studies and diagnosis (see review of his very different talk last year at ECA Dublin ECA Newsletter 26 and on this meeting report). Malcolm showed the effectiveness of sex-sorting bovine sperm by flow cytometry using his X and Y chromosome paints, and then went back to several examples showing a range of farm animals with chromosome aberrations: 2n=55, XXY rams and XX/XY freemartin sheep as a consequence of development of mixed-sex twins. His comparisons with equivalent cases in human were very relevant to the development of the field. Malcolm then explained our current understanding of the chromosome biology of the horse x donkey hybrid, the mule, and the reciprocal cross, the hinny. Both hybrids have 2n=63 chromosomes, with one set of 32 from the horse and the other of 31 from the donkey (many new results built on Yang et al. Chromosome Research 2004). Following the exceptionally clear demonstration of evolutionary conservation of chromosomal segments between horse and donkey, the results showed how whole horse sets can segregate at meiosis to give occasional fertility in the hybrids species.

Bhanu Chowdhary styled his talk as being ‘reflective’ in nature, having moved to a new senior University administrative position in Qatar earlier in the year from a long career in Texas. He discussed how chromosomes and genome analysis are transforming the landscape of cytogenetic applications in the 21st century: work from chromosomes initially was laying the foundation for maps of domestic animal karyotypes, then comparative cytogenomics and now sequencing … and moving on to metabolomics, transcriptomics and proteomics. Bhanu discussed achievements from genome structure studies from the chromosome to DNA which lead to causes of diseases being deciphered and the role of single gene mutations; a challenge remains in defining multifactorial traits. He ended by discussing some of the challenges in food security, illustrating the selection of near-‘monstrous’ animals with extreme hind-quarters, udders, wool or egg production.

Jerry Taylor asks a question at a session cytogenetics Colloquium Ischia Italy
Jerry Taylor asks a question at a session of the cytogenetics colloquium, Ischia Italy

The final talk of the opening session came from Jerry Taylor (Texas A&M University): he started with the admission that he is an NGS (next generation sequencing) addict – about 10% of the audience also admitted to the addiction, although he didn’t ask the reciprocal question! For a typical mammalian genome of about 3,000 Mbp, he suggested that at least 23x sequence coverage was required to give suitably robust results. Although 10x sequence coverage would be expected to miss a very low 10^-6 proportion of the genome in the sequence, experience shows that some 10% of genes are missed because of non-random sequence coverage. At 30x coverage, only 0.5% of a genome has no coverage. Moving on to his own work, Jerry’s group has now sequenced 65 dogs, and worldwide there are about 106 sequences; individual dogs are more variable than individual humans. NGS sequencing and comparative analysis has proved phenomenally powerful – his lab has found 8 mutations causing dog diseases in last year, all as loss of function alleles. The sequencing approach is finding deletions as well as SNPs, and he showed one dog disease is a 117bp deletion, again a loss of function alleles. With previous approaches, it was half of a career to find a disease locus, and even fine-mapping finds large genome regions – millions of base pairs and a few dozen candidate genes!

Moving on to cattle, Jerry showed some interesting comparisons between genome sequences of Hereford cattle – brown with white faces – and the Black Angus breed. Evidence for selective sweeps was clear in Herefords, leading to regions where all loci are fixed so there is no variability. Comparing Angus and Hereford, one of the comparisons he showed had hundreds of SNPs and 33 small indels that were fixed for different alleles between Angus and Hereford, all in non-coding regions. The white-face character arises from a duplication, probably disrupting a long-range expression enhancer. Different data types are important for the analyses: from a long term pedigree with 19 generations back to the mid-1950s, to no less than 100 Terabytes of whole-genome sequencing data. In the cattle, 10% of all pregnancies are lost because of homozygosity for loss of function alleles. Once these LoF alleles are found, there will be an assay for c. 2000 alleles, and 10,000 animals will be genotyped to find how many loci are never homozygous because of lethality.

Another theme of the conference was the clinical genetics of sexual development: how does the undifferentiated gonad develop, and what can go wrong? The topic was overviewed by Pietro Parma (Milan, Italy): the consequences of SRY abnormalities, the universal effect of Sox9 and R-spondin1 in sex-reversal were given. Detailed examples including horses and dogs were described in following talks and posters.

Mariano Rocchi, board member of ECA from Bari, Italy, was introduced as the father of neocentromeres. He presented the evolutionary history of chromosomes from the point of view of the movement of the centromere, either through a pericentric inversions, or activation of a neocentromere. Another section of his talk covered a theme running through many of the sessions at the meeting: the need for refinement and correction of genome assemblies from sequence data to use cytogenetic approaches.  He showed how BAC-FISH is solving frequent assembly problems in the bovine genome.

Raquel Chaves UTAD talked about cat and dog repetitive DNA but is here seen on the Conference boat trip!
Raquel Chaves UTAD talked about cat and dog repetitive DNA but is here seen on the Conference boat trip!

Repetitive DNA sequences are a major fraction of most genomes, and lead to most of the difficulties in assembly of sequences. Raquel Chaves (Vila Real, Portugal, and our collaborator in some other projects) presented exciting results with satellite DNA from Peromyscus and other Cricetidae species, showing how it evolves by copy number variation. She noted where new sequencing technologies are valuable (particularly compared to PCR with specific primers) to show the complexity of the sequence variation and organization of these satellites. The second part of her talk focussed on a sub-telomeric satellite (rather than the very frequent centromeric satellites in mammals) from cats. Interestingly, these were transcribed and could be seen at different places in the nucleus – sometimes widely distributed, sometimes more nucleolus related, and transcription was linked to cell cycle with immune-markers. Cellular stress could increase, or in the case of starvation decrease, transcription, while 5-Azacytidine to modify DNA methylation had effects after treatment, but also suggested very complex regulation.

My own talk (Pat Heslop-Harrison, Leicester, UK) focussed on comparative evolution of the satellite DNA sequences at the centromeres of cows, goats, sheep and pigs, and showed aspects of the organization and methylation at meiosis in pigs. Another talk in this session by Martine Yerle-Bouissou (Toulouse, France) advanced understanding of the 3D remodelling of chromatin as a determinant for genomic regulation, pinpointing the major role of 3D nuclear organization. Looking at chromosome territories, centromeres and telomeres, they were able to understand the complex organization of the MHC major histocompatibility complex using BAC-FISH.

Feng-Tang Yang (Cambridge, UK) continued the discussion of pig chromosomes, with a focus on the challenge of FISHing with probes containing repeats, and the use of molecular combing as a superior technology for ordering and gap filling in the Yp arm.

Jiri Rubes, Veterinary Research Institute, Brno
Jiri Rubes, Veterinary Research Institute, Brno discussing zoo animal cytogenetics at coffee time

Jiri Rubes (Brno, Czech Republic) gave a wide ranging talk on cytogenetic abnormalities in zoo animals – critical work to ensure captive animals remain healthy and contribution to conservation programmes. His lab has investigated more than 1000 animals from Bovids and 200 Equidae over last 5 years, finding numerous chromosome aberrations.

Darren Griffin finishing his talk on raptor chromosomes in Ischia
Darren Griffin finishing his talk on raptor chromosomes in Ischia

The final series of talks extended discussion of evolutionary cytogenetics in a very broad range of vertebrates and invertebrates. Darren Griffin (Kent, UK) presented a synthesis of his work on the avian genome, as a very fragmented genome with many microchromosomes; this presentation complemented a special issue of Chromosome Research which we had in the conference folder. Most of the 1000 avian karyotypes are partial because of number of microchromosomes: only the chicken has a full karyotype, but 63% of the species are approximately 2n=80.  Once more, classical karyotyping is undergoing a revival because of the problems of fragmented genome assemblies from sequencing data, where about 60 bird genomes are soon to be available. Darren’s work has also many cross-species of copy number variations, CNVs – 790 apparent CNVs within 135 unique regions, although there seemed to be no more CNVs in species with more rearranged karyotypes. Another section of his talk focussed on these evolutionary breakpoints based on homologous syntenic regions, before consideration of the ancestral karyotype spanning dinosaurs, snakes and other reptiles, and birds. So the next remake of Jurassic Park will include Saurian chromosomes!

The considerable progress in understanding chromosome evolution and speciation was emphasized in a series of talks. Valerie Fillon (Catanet-Tolosan, France) extended the evolutionary discussion of birds by comparing duck (2n=80) and chicken (2n=78). Since separation of the species 80 million years ago, there have been many complex intrachromosomal rearrangements including inversions, translocations and smaller or larger rearrangements that can be demonstrated by FISH, although only the single chromosome fission with chicken GGA4 splitting in duck. Elena Giulotto (Pavia, Italy) showed her substantial progress towards understanding the dynamic evolution of centromeres in the horse genus Equus, where chromosome number varies from 2n=66, to 2n=64 in horse, down to 46, 44 and even 2n=32 in Hartman zebra, despite the fact that many species can still interbreed. Satellite DNA was very helpful as a probe to show features of chromosome rearrangement and the frequent repositioning of centromeres, and she has developed a model for centromere repositioning during evolution. This could explainin the localization of sat DNA in the equids, including the stable centromeres without satellites and the ‘sliding’ positions of centomeres, concluding that DNA sequence has no role in centromere identity.

Animal cytogeneticists at coffee time
Animal cytogeneticists at coffee time

Insight into invertebrate chromosomes and genomics came from several talks: Catherine Ozouf-Costaz (Paris) noted how fish cytogeneticists need to be clever! There are impressive cytogenomic advances in teleost fish recently, although only 15% have been karyotyped, and she noted how important it is to retain voucher specimens because of the uncertain taxonomy. Some 72% of species have only a single pair of 45S rDNA loci, although 5S rRNA gene mapping shows high variability at molecular level but low variability at chromosome level. Telomeric repeats (TTAGGG) show frequent occurrence at intercalary as well as the terminal positions on chromosomes. Other exciting work covered lampreys, Arctic and Antarctic species, and microspeciation in fishes, with excursions into bivalve and crustacean cytogenetics.

The complete book of abstracts from the meeting is available from the Chromosome Research website, and giving summaries of many other important results I have not been able to report here. Overall, the conference showed the significant impact that whole-genome sequencing is having on cytogenetics, but emphasized how important study of the chromosomes is to understanding the most significant evolutionary processes – including chromosome fusion, fission, inversion, and centromere repositioning, as well leading to unique understanding of the role of duplications and deletions of different classes of sequences.

Poster Prize winner Alsu Saifitdinova with Valerie Fillon
Poster Prize winner Alsu Saifitdinova with Valerie Fillon

The posters were notable not only for the excellent science but also the range of species and techniques used – including bats, chickens, rodents and various invertebrates that are models, domesticated or wild species, and using methods of electron and light microscopy and sequencing and bioinformatics.

The following outstanding presenting authors, from a range of excellent posters, were awarded Colloquium Poster Prizes:

A. Saifitdinova (St Petersberg, Russia) P15. Cytogenetic description of chicken microchromosomes at the lampbrush phase

B. Piccinni  (Salento, Italy) P22. Genomic analysis of the ovine T cell receptor alfa/delta (TRA/TRD) locus deduced by comparative and expression analyses

T.F.A. Ribas (Belem, Brazil) P30. Phylogenetic reconstruction in Phyllostomini tribe (Chiroptera, Phyllostomidae) based on cross-species chromosome painting

L. Belkadi (Paris, France) P44. Bursts of retrotransposons and extensive chromosomal repatterning within the Antarctic teleost fish species flock Trematominae.

C. Araya-Jaime (Botucatu, Brazil) P40. Chromosomal mapping of 5 classes of repetitive DNAs in three species of the genus Eigenmannia (Gymnotiformes, Sternopygidae).


The Chromosome Research poster prize was awarded to M.I. Rahn (Buenos Aires, Argentina) P25. Synaptic behavior and chromatin remodeling of the multiple sex chromosomes in bats

PHH is grateful the European Cytogeneticists Association (ECA, in conjunction with the Annual General Meeting and developing plans for the 10th European Cytogenetics Conference, 2015, in Strasbourg) and the journal Chromosome Research for support of my attendance at the Colloquium.

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