Molecular cytogenetics research group October 2014

MolCytGroupSept2014_PHH7675bAt our lab. meeting a couple of weeks ago, we were happy to welcome two new PhD students to the group. Here we all are in the lobby of our Adrian Building. We are from six countries, and work on a diverse range of molecular cytogenetics projects.

From left to right, left of DNA molecule: Amal Alyamani (Wheat repetitive DNA), Ramesh Patel (Technical), Chetan Patokar (Wheat-Thinopyrum introgression), Rubar Salih (Taraxacum diversity), Pat Heslop-Harrison, Juceli Gouveia (Fish microsatellite organization) ; Sarbast Mustafa (Sheep and bovid satellite DNA diversity). Right of DNA molecule: Trude Schwarzacher, Jotyar Mohammed (Aubrieta biodiversity), Khaloud Alarjani (Brassica repetitive DNA), Stuart Desjardins (Fallopia/Knotweed relationships), Nauf Alsayid (Saffron and Crocus diversity), Fabiola Santos Carvalho (Brachiaria forage grass molecular cytogenetics; her picture is inserted). Adel Sepsi (Centromeres and epigenetics in wheat introgression lines).

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Chromosomal evolution in Brachiaria forage grasses with Fabiola Carvalho Santos

Fabiola Santos overlooking Brachiaria trials in Brazil

Fabiola Santos overlooking Brachiaria trials in Brazil

Fabiola Santos from University of Londrina, Brazil, is working on the Chromosomal evolution and the organization of repetitive DNA sequences in diploid and polyploid Brachiaria forage grasses in the molecular cytogenetics group in LeicesterBrachiaria is most important cultivated forage grass genus in Brazil (with billion-dollar production), and the genus includes diploid and polyploid species with similar and small chromosomes and basic numbers of x=6, x=7 and x=9. Many are apomictic and may multiply vegetatively. Fabiola is studying the genome structure and the monoploid complement to provide information about the origins of the polyploids and structure of the genome. In particular, using bioinformatic and molecular cytogenetic methods, she is exploiting rDNA, microsatellites, tandem repeats and transposable elements to characterize major genomic components, and to identify chromosomes by in situ hybridization. Questions include What is the monoploid complement – x number – for Brachiara? What is the distribution of retroelements and microsatellites across the genomes? Can we develop probes that allow identification of many individual genomes and chromosome types – with either localized probes or whole-chromosome paints?

Brachiaria bryzantha chromosomes (2n=4x=36) stained  blue with DAPI and showing some chromosomes with less labelling with a retroelement probe.

Brachiaria bryzantha chromosomes (2n=4x=36) stained blue with DAPI and showing some chromosomes with less labelling with a retroelement probe.

As well as developing karyotypes and understanding processes of genome evolution and speciation, the results will assist breeders in characterization of interspecific hybrids.

Her preliminary results are presented at the Plant Molecular Cytogenetics in Genomic and Postgenomic Era Conference in Katowice, Poland, in September 2014 in a poster by Fabíola Carvalho Santos, André Luiz Laforga Vanzela, Trude Schwarzacher and Pat Heslop-Harrison.

Fabiola is based in the Department of Biology Science, University of Londrina, Londrina, PR, Brazil. email: fabiolacs1786(a)gmail(dot)com

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George Fraser (1955-2014) Professor of Detector Physics and his biology

Professor George Fraser (1955-2014)

Professor George Fraser (1955-2014)

My colleagues and I were devastated by the news of the untimely death of our collaborator and friend George Fraser (22 July 1955 — 18 March 2014). George had a unique combination of vision of the applications of detector physics, knowledge from space research, optics, and electronics which is a huge loss to the whole research and imaging community. He was Professor of Detector Physics and Director of the Space Research Centre of the University of Leicester.

I had the privilege of working with George from soon after I joined the University in 2000. We were interested in spectral imaging and quantitative fluorescence microscopy. Within a short time of our first meeting, it was clear that the detectors he was working with for space applications could address the problems we were facing in microscope imaging. These included spectral imaging, to get away from the need to put filters or gratings in front of fundamentally monochromatic detectors, and the quantification of signal. Our discussions soon led to collaboration exploiting his knowledge of novel detector technology, and after proof-of-principle experiments with George at European Space Agency Technology Centre ESTEC, we wrote two manuscripts and a patent (linked below).

George and STJ drawings

George and STJ drawings

Moving beyond the preliminary experiments, it became clear there were additional advantages in huge signal-to-noise ratios, background rejection, and novel ways the detectors could be used with the additional dimension of photon arrival time.

Another strength of George soon became apparent in the wish to exploit the technology. In October 2004, we founded a company, BioAstral Limited, to develop the technology for a broad range of applications in biology. As we wrote, the STJ cryogenic detector is 1000 times more powerful at detecting fluorescence in biological assays than current technology, and it is unique in giving the colours of photons arriving without resort to filters, gratings or other spectroscopy.

George Fraser, Pat Heslop-Harrison, Andrew Holland and Trude Schwarzacher - Inventors on the STJ patent

George Fraser, Pat Heslop-Harrison, Andrew Holland and Trude Schwarzacher – Inventors on the STJs in biology patent

George had amazingly broad interests and unique insight into applications of his research, in technical and commercial terms – he really knew what ‘impact’ was. As such, he was looking for ways that Space science, and particularly his expertise in imaging, could be developed into a commercial product. At the time of his death, he was a Director of three commercial limited companies, BioAstral, Spectral ID and Gamma Technologies, as well as a Director of the National Space Centre’s NSSC Operations.

The cryostat/refrigerator/cooler for the optical STJ operating at 280mK in Leicester

The cryostat/refrigerator/cooler for the optical STJ operating at 280mK in Leicester

Developing the imaging technology where I was involved had formidable technical challenges with respect to obtaining the temperatures required for operation, building the electronics and engineering both the optical and electronic outputs, and George was up to continuous developments in this direction, towards having our own detector connected to a microscope in Leicester. Work he led ranged from getting the temperatures of a few hundred millikelvin without needing hundreds of litres of liquid helium, through to trapping of the earth’s magnetic field in the superconducting junctions and googlies like Oxford Instruments supplying US 120V equipment with UK 240V connectors which slowly blew up. Each one of challenges George could overcome with innovative solution, but sadly we never truely got beyond ‘first light’ in telescope terminology with the system in Leicester.

In other work in biology, George was able to use his insight in mathematical analysis of photon colour based on novel understanding of the optical properties of fluorochromes used in biology. This lead to some really interesting work and clearly could have been exploited for improved extraction of data from any multifluorescence assays used in biology. While there was substantial work involving a number of students, these ideas were only published in a patent (see below).

A huge regret in the work with George was that we never succeeded in obtaining any significant funding to develop the detector technology to the point it could be used routinely in biology. All our work was done with shoe-string funding, and we all were putting giant amounts of effort into writing applications and discussing the implications and applications, but it was all regarded as too early stage or too late stage.

I will miss my regular and wide ranging discussions with George on imaging and analysis. I remain in no doubt that cryogenic detectors have a major role in biology, as well as space science, in the future. Their development has been set back enormously by George’s sudden death. I hope that one of the many memorials to him will be the future widespread application and exploitation of STJs and TESs.

This in memoriam note is the basis of my overview of George’s work in biology for the memorial celebration held at the University of Leicester for George Fraser on 5th September 2014

Pat Heslop-Harrison.

I have published two papers jointly with George over the years:
245. Fraser GW, Heslop-Harrison JS, Schwarzacher T, Verhoeve P, Peacock A, Smith SJ. 2006. Optical fluorescence of biological samples using STJs. Nuclear Instruments & Methods in Physics Research Section A-Accelerators Spectrometers Detectors and Associated Equipment 559(2): 782-784. doi:10.1016/j.nima.2005.12.136

226. Fraser GW, Heslop-Harrison JS, Schwarzacher T, Holland AD, Verhoeve P, Peacock A. 2003. Detection of multiple fluorescent labels using superconducting tunnel junction (STJ) detectors. Review of Scientific Instruments 74 (9): 4140-4144. DOI: 10.1063/1.1599059

Another paper was in an advanced draft at the time of George’s death:

Labelled cell images detected by an STJ array on a fluoresence microscope

Labelled cell images detected by an STJ array on a fluoresence microscope

G. Torricelli, Trude Schwarzacher, Peter Verhoeve, Didier Martin, J. S. (Pat) Heslop-Harrison, George W. Fraser et al. Hyperspectral imaging of biological samples using a superconducting tunnel junction (STJ) camera
We are now working to complete this work.

We have a patent issued for use of cryogenic detectors – either the fast-responding STJ superconducting tunnel junctions, or slower TES, transition edge sensors.

Fraser GW, Heslop-Harrison JS, Holland AD, Schwarzacher T (unordered) 2003. Detection of the energy of photons from biological assays. Patent. September 2001, published 20th March 2003 under publication number WO 03/0233 76, application number is PCT/GB02/04019

A second patent on the quantitative analysis of biological fluorochromes was also granted: Fluorescence labelling Fraser GW and Ray DJM. WO 2008081203 A2 “This invention generally relates to techniques for fluorescence labelling, and to methods, apparatus and computer program code for processing fluorescence signal data.”

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An Overview of Peanut Genome Structure

Peanut genome structure: a tetraploid with many repetitive elements

Peanut genome structure: a tetraploid with many repetitive elements

308. DJ Bertioli, ACG Araujo, S Nielen, P Heslop-Harrison, PM Guimarães, T Schwarzacher, S Isobe, K Shirasawa. 2014. An overview of peanut genome structure. Chapter 6 in Genetics, Genomics and Breeding of Peanuts. Eds Nalini Mallikarjuna, Rajeev K. Varshney. Peanut genome structure manuscript Author Version pp. 114-138. CRC Press, Florida.

ABSTRACT Cultivated peanut (Arachis hypogaea L.) is a Papilionoid grain legume crop, important throughout the tropics. It is an allotetraploid of recent origin with an AB type genome (2n= 4x= 40) and has very low DNA polymorphism, a characteristic that has hampered genetic studies. The A and B genomes are of similar size and are composed mostly of metacentric chromosomes. The A genome is characterized by a pair of small chromosomes and the presence of strong centromeric heterochromatic bands, in contrast, B chromosomes are all of similar size and have much weaker centromeric bands. The genome of peanut is estimated at about 2.8 Gb and with a high repetitive DNA content. Its most probable diploid ancestors are A. duranensis and A. ipaënsis, donors of the A and B genomes, respectively. These two subgenomes diverged from a common ancestor about three and a half million years ago, more recently than the subgenomes of cotton or soybean. Consequently, homeologous A and B genic sequences have very high sequence identity. Genetically, cultivated peanut behaves as a diploid. The two subgenomes have very high genetic synteny, and do not appear to have undergone major structural rearrangements after polyploidization. Indeed, the peanut subgenomes even have detectable genetic synteny with other legumes that diverged during evolution about 55 million years ago. The patterns of synteny indicate that the A and B genomes are diploidized, and that gene-space is likely to be ordered into about ten conserved blocks. In contrast to their conserved genetic synteny, the repetitive DNA components of the subgenomes are very significantly diverged. This may be substantially explained by the activity of a few retrotransposons since the time of genome divergence. In addition, the peanut genome harbors many miniature inverted-repeat transposable elements that have been active since polyploidization. This activity has probably contributed to the phenotypic variability of peanut.
Genetics, Genomics and Breeding of PeanutsLink to book on publishers website.
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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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Sugar cane and characterization of Saccharum hybrids by molecular cytogenetics with Natalia Melloni

Natália Melloni from UNESP, Brazil, fixing sugar cane root tips

Natália Melloni from UNESP, Brazil, fixing sugar cane root tips

Natália Melloni from UNESP and IAC, Brazil, has been characterizing Saccharum sugar cane hybrids in a collaboration with the molecular cytogenetics group.

The title of the project is “Characterization of interspecific hybrids (Saccharum spp x Saccharum spontaneum) by molecular cytogenetics” or “Caracterização de híbridos interespecíficos (Saccharum spp x Saccharum spontaneum ) por citogenética molecular”, and involves several collaborators from the Faculdade de Ciências Agrárias e Veterinárias, UNESP, Jaboticabal, Sao Paulo and Centro Avançado da Pesquisa Tecnológica do Agronegócio de Cana, IAC/Apta, Ribeirão Preto, SP, Brazil as well as MNG (Natalia) Melloni: Melloni, MLG; Rossini, L; Creste, S; Xavier, MA; Landell, MGA.

Natalia first tested the technique on normal sugarcane (Saccharum officinarum) from our Botanic Garden and then analysed 5 of her hybrids (Saccharum ssp. x S. spontaenum), clone 221, clone 292, clone 297, clone 303 and clone 316.  She was able to differentiate the chromosomes originating from either parent by genomic FISH and could show that both contribute to the hybrid in the predicted proportions. Natalia has obtained a good number of very good images of the chromosomes of Sacharrum that will be able to be published. However, sugarcane chromosomes are extremely small and plentiful (2n=120-130) and therefore detailed cytogenetic analysis and estimation of any recombinant chromosomes is not possible with the genomic probes. Further the hybrids analysed are F1 hybrids and have not gone through meiosis yet and no recombination was expected. However it is important to establish the true hybrid nature of her plants before directed crossing experiments. We discussed possible bioinformatic approaches which may make probes with enhanced genome-specificity as genomic DNA sequences may become available.

An abstract of a talk and future work is below:

Melloni, MNG¹; Melloni; MLG¹, Rossini, L ¹,2; Creste, S²; Xavier, M.A², Landell, MGA2, Heslop-Harrison, JS, Schwarzacher, T, Bailey, JP

¹ Faculdade de Ciências Agrárias e Veterinárias, UNESP, Jaboticabal, SP.

² Centro Avançado da Pesquisa Tecnológica do Agronegócio de Cana, IAC/Apta, Ribeirão Preto, SP.

Financial support for the work came from CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil

Sugarcane is an important crop worldwide due to sugar, ethanol and more recently biomass production. Modern sugarcane varieties, Saccharum spp are vigorous hybrids originating mainly from interspecific crosses between S. officinarum (a high sugar content species) and S. spontaneum (low sugar content and high fiber) followed by several backcrosses to S. officinarum to recover sucrose content. During backcrossing, the sugarcane varieties have lost important features related to high biomass and fiber production. Introgression programs focused on biomass and fibre have used crosses between sugarcane commercial varieties and S. spontaneum accessions. The present work aimed to better understand the processes of  n+n or 2n+n transmission in a group of individual “hybrids” derived from a cross between the commercial cultivar RB855465 (2n=100-130) and S. spontaneum (GlagaH; 2n=112) and to investigate the contribution of S. spontaneum in the genome of these hybrids using genomic in situ hybridization (GISH) to chromosome preparations. After the confirmation of the hybrid nature by molecular markers (microsatellites), roots collected from germinated buds (BOD at 35ºC) were immersed for 4 hours in 0.04% 8-hydroxyquinoline before fixation. Total genomic DNA was extracted from S. officinarum leaves (Green German) and S. spontaneum (GlagaH) and labelled for GISH. The chromosome number was 2n= 100-118, confirming the haploid nature of gametes and the n+n transmission of parents. The genomic DNA probes labelled all the chromosomes and gave weak discrimination of two sets of chromosomes. The S. spontaneum probe labelled the centromeric regions most strongly, while the S. officinarum probe was more dispersed along the length of the chromosomes. In the accessions, about 60% of chromosomes labelled with the S. spontaneum probe and 40% with S. officinarum, supporting the hybrid nature of the S. officinarum parent; no recombinant chromosomes were detected. Given the differential labelling patterns, the method is likely to be valuable for defining the chromosome complement of sugar cane hybrids and derivative lines. More genome-specific probes may be developed with whole genome sequence information for use in identifying chromosomes in hybrids and helping target development of new lines.



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The origin of an old, resource-efficient crop, Broomcorn millet or Panicum miliaceum

Panicum: the origin of tetraploid proso or broomcorn millet

Panicum: the origin of tetraploid proso or broomcorn millet

307. Hunt HV, Badakshi F, Romanova O, Howe CJ, Jones M, Heslop-Harrison JS. 2014. Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P. miliaceum. Journal of Experimental Botany 65 (12), 3165-3175.  DOI:10.1093/jxb/eru161 . (Link to local copy J. Exp. Bot.-2014-Hunt-3165-75)

Panicum miliaceum (broomcorn millet) is a tetraploid cereal which was among the first domesticated crops, but is now a minor crop despite its high water use efficiency. The ancestors of the species have not been determined; we aimed to identify likely candidates within the genus, where phylogenies are poorly resolved. Nuclear and chloroplast DNA sequences from P. miliaceum and a range of diploid and tetraploid relatives were used to develop phylogenies of the diploid and tetraploid species. Chromosomal in situ hybridization with genomic DNA as a probe was used to characterize the genomes in the tetraploid P. miliaceum and a tetraploid accession of P. repens. In situ hybridization showed that half the chromosomes of P. miliaceum hybridized more strongly with labelled genomic DNA from P. capillare, and half with labelled DNA from P. repens. Genomic DNA probes differentiated two sets of 18 chromosomes in the tetraploid P. repens. Our phylogenetic data support the allotetraploid origin of P. miliaceum, with the maternal ancestor being P. capillare (or a close relative) and the other genome being shared with P. repens. Our P. repens accession was also an allotetraploid with two dissimilar but closely related genomes, the maternal genome being similar to P. sumatrense. Further collection of Panicum species, particularly from the Old World, is required. It is important to identify why the water-efficient P. miliaceum is now of minimal importance in agriculture, and it may be valuable to exploit the diversity in the species and its ancestors.

(Link to local copy J. Exp. Bot.-2014-Hunt-3165-75) DOI :10.1093/jxb/eru161.

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