ImmunoFISH – Simultaneous visualisation of proteins and DNA sequences gives insight into meiotic processes in nuclei of grasses

Sepsi A, Fábián A, Jäger K, Heslop-Harrison JS, Schwarzacher T. ImmunoFISH: simultaneous visualisation of proteins and DNA sequences gives insight into meiotic processes in nuclei of grasses. Frontiers in Plant Science. 9: 1193. https://doi.org/10.3389/fpls.2018.01193

Combined Immunolabelling and In Situ Hybridization FISH. Sepsi et al. 2018. Frontiers Plant Sci 9: article 1193

Combined immunolabelling and in situ hybridization FISH. Sepsi et al. 2018. Frontiers Plant Sci 9: article 1193

ImmunoFISH is a method combining immunolabelling (IL) with fluorescent in situ hybridisation (FISH) to simultaneously detect the nuclear distribution of proteins and specific DNA sequences within chromosomes. This approach is particularly important when analysing meiotic cell division where morphogenesis of individual proteins follows stage-specific changes and is accompanied by a noticeable chromatin dynamism. The method presented here is simple and provides reliable results of high quality signal, low background staining and can be completed within 2 days following preparation. Conventional widefield epifluorescent or laser scanning microscopy can be used for high resolution and three-dimensional analysis. Fixation and preparation techniques were optimised to best preserve nuclear morphology and protein epitopes without the need for any antigen retrieval. Preparation of plant material involved short cross-linking fixation of meiotic tissues with paraformaldehyde (PFA) followed by enzyme digestion and slide-mounting. In order to avoid rapid sample degradation typical of shortly fixed plant materials, and to be able to perform IL later, slides were snap-frozen and stored at -80C. Ultra-freezing produced a remarkable degree of structural preservation for up to 12 months, whereby sample quality was similar to that of fresh material. Harsh chemicals and sample dehydration were avoided throughout the procedure and permeability was ensured by a 0.1–0.3% detergent treatment. The ImmunoFISH method was developed specifically for studying meiosis in Triticeae, but should also be applicable to other grass and plant species.

 

Sepsi A, Fábián A, Jäger K, Heslop-Harrison JS, Schwarzacher T. ImmunoFISH: simultaneous visualisation of proteins and DNA sequences gives insight into meiotic processes in nuclei of grasses. Frontiers in Plant Science. 9: 1193. https://doi.org/10.3389/fpls.2018.01193

 

 

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An Immortalized Genetic Mapping Population for Perennial Ryegrass: A Resource for Phenotyping and Complex Trait Mapping

Velmurugan J, Milbourne D, Connolly V, Heslop-Harrison JS, Anhalt UC, Lynch MB, Barth S. 2018. An immortalized genetic mapping population for perennial ryegrass: a resource for phenotyping and complex trait mapping. Frontiers in Plant Science 9: article 717https://doi.org/10.3389/fpls.2018.00717

Lolium Genetic Map and RI lines showing population structure. Velmurugan et al. 2018. Frontiers in Plant Science 9: article 717

Lolium Genetic Map and RI lines showing population structure. Velmurugan et al. 2018. Frontiers in Plant Science 9: article 717

To address the lack of a truly portable, universal reference mapping population
for perennial ryegrass, we have been developing a recombinant inbred line (RIL)
mapping population of perennial ryegrass derived via single seed descent from a
well-characterized F2 mapping population based on genetically distinct inbred parents
in which the natural self-incompatibility (SI) system of perennial ryegrass has been
overcome. We examined whether it is possible to create a genotyping by sequencing
(GBS) based genetic linkage map in a small population of the F6 generation of this
population. We used 41 F6 genotypes for GBS with PstI/MspI-based libraries. We
successfully developed a genetic linkage map comprising 6074 SNP markers, placing a
further 22080 presence and absence variation (PAV) markers on the map. We examined
the resulting genetic map for general and RIL specific features. Overall segregation
distortion levels were similar to those experienced in the F2 generation, but segregation
distortion was reduced on linkage group 6 and increased on linkage group 7. Residual
heterozygosity in the F6 generation was observed at a level of 5.4%. There was a high
proportion of chromosomes (30%) exhibiting the intact haplotype of the original inbred
parents of the F1 genotype from which the population is derived, pointing to a tendency
for chromosomes to assort without recombining. This could affect the applicability
of these lines and might make them more suitable for situations where repressed
recombination is an advantage. Inter- and intra-chromosomal linkage disequilibrium
(LD) analysis suggested that the map order was robust. We conclude that this RIL
population, and subsequent F7 and F8 generations will be useful for genetic analysis
and phenotyping of agronomic and biological important traits in perennial ryegrass.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991167/pdf/fpls-09-00717.pdf

https://doi.org/10.3389/fpls.2018.00717

Keywords: perennial ryegrass, Lolium perenne, recombinant inbred lines (RIL), genotyping by sequencing, mapping population, phenotyping

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Herbert Macgregor 1933-2018: a personal tribute

Topsham Church and Professor Herbert Macgregor's Memorial Service

Topsham Church and the Service of Thanskgiving for the Life of Professor Herbert Macgregor

My Tribute to Professor Herbert Macgregor (22nd April 1933 – 22nd July 2018) delivered at his Thanksgiving Service on 13th August 2018

I am humbled to be here today to pay tribute to the wonderful scientist, leader and mentor, Herbert Macgregor. As a fellow cytogeneticist, Herbert has been right at the top of my field, and despite the great sadness, I am happy to be able to give you some thoughts about his remarkable and productive life. I first came across Herbert’s name in my earliest undergraduate studies with other cytogeneticists, Carl Swanson and Hugh Rees, learning of Herbert’s discovery (with Mick Callan) of the fact that each of the chromosomes within a cell nucleus – 46 of them in the case of human – contained a single DNA molecule, 100s of millions of DNA bases long, from end to end (Callan and Macgregor, 1958). Today, this is such a tenet of cell biology and genomics that it is accepted as ‘obvious’: the importance of the result supported by detailed experimental work stands equal with the other key discoveries of the late 1950s and 1960s, including the DNA helix, the genetic code, transcription, and semi-conservative replication of DNA. Herbert was a pioneer with techniques, using them and developing model systems to address many novel questions throughout his career. His book from the early 1980s, “Working with Animal Chromosomes” is still the standard reference for chromosome preparations and regularly used in my lab, while his 1993 volume, Introduction to Cytogenetics, is another standard science for interpreting chromosome form and behaviour, and appreciating its evolutionary significance.

I first met Herbert at a chromosome conference in 1982 organized in Lübeck, Germany. Herbert’s talk was a highspot of the meeting – one of those annotated with three stars in my programme. Like so many of Herbert’s papers, the title is as thoughtful and current now as when the paper was presented and written up, “The evolutionary consequences of major genomic changes”: remember this was long before the science of genomics was the huge industry that it has become. Other papers had titles such as “High resolution map” or “Application of the technique”, or discussed karyotypes and chromosome morphology. Herbert’s paper, based on his own detailed investigations of the chromosomes in one of his favoured groups, the newts and other amphibians, makes conclusions and generates ideas that are still current and important today.

Two years later, Herbert and Alma organized an important meeting of the British Society of Developmental Biology, BSDB, in Leicester, where Herbert was Professor and Head of the Department of Zoology from 1970. This was one of my first conference talks, and I remember well the exceptional organization and range of exciting talks on the topic “Genes, chromosomes and computer models in developmental biology”. At that time 34  years ago, “Computer models”  were unheard of in chromosome biology, but the contents of the volume are truly visionary. Herbert and Alma’s words in the introduction, “the frontier of this symposium lies between the biologists and computer scientists, between biology and biochemistry and mathematics and modelling” well summarize the range of Herbert’s research and its implications for the rest of his career. This 1984 meeting was my first visit to Leicester where I am now based, and there were some strange events around the conference. I remember ending up in a pub with a number of the most notable conference participants, including Adrian Bird and Chris Bostock, and witnessing still the most vicious fight I have ever seen, as well as one or two unusual conference participants!

I continued to follow with much excitement Herbert’s work, now linking gene expression with its physical manifestation as loops coming out from a special type of chromosome – lampbrush chromosomes – in amphibians and birds. This research on the arrangement and expression of DNA sequences in the genomes had major implications for molecular biology and the functioning of cell nuclei, as well as for evolution and developmental biology. Once again, this research is now part of the unquestioned canon of scientific knowledge. All through his work, from his very first papers in the late 1950s, Herbert was able to integrate microscopy with molecular biology – something that is today even more important than it was 50 years ago.

I got to know Herbert much better from the early 1990s, when he invited me to join him in a new publishing venture, setting up the Journal Chromosome Research, now in its 25th successful year. With Herbert’s vision, it has gained a distinctive niche in a publishing area, long before the huge expansion in numbers of publications the last decade. I gained enormously from seeing his firm but helpful interactions with the series of publishers we worked through, and carefully thought-through handling of the interactions with authors and editors to build the reputation of the journal. We were now in close contact, not only with publishing but with respect to our research and the chromosome research environment. So I discussed with him about 1999 that my then institute was in an uncomfortable state of upheaval, and about any academic positions coming up? Retiring around that time, but still maintaining his lab., he asked the right people and soon after, a rather specialized advertisement appeared in a newspaper, sandwiched between adverts for the chief fire officer for B&Q (a chain of UK DIY shops), and head groundsman for the Leicester City Football Club. So I moved to Leicester, and his wise advice was so valuable to me. Most notably, Herbert valued the exceptional people he worked with, particularly among the support staff. He invariably appreciated their immense contributions, showing the importance of their roles, and the impact they could have. In this way, he was able to build an extraordinary and productive world-class Department, and I saw the remaining atmosphere he fostered in Leicester. Within the environment he fostered in his Department, he had no truck with nonsense from administrators and senior colleagues, and was forthright in his robust views. I should also add that he held the most wonderful Christmas parties, with several dozen people from the wider Department.

Herbert cared deeply about his teaching and undergraduates and gave me extremely valuable advice as I took over in teaching some of the introductory genetics and biology courses. His lecturing style was immensely approachable but still challenging, and I was not surprised to hear he continued outreach and learning activities through U3A here in Exeter. Among other things, he maintained a remarkable website here at the University of Exeter on lampbrush chromosomes, providing a resource for all – not least with its progressive CC-BY open accessibility without copyright.

Personally, it was always wonderful and memorable to meet Herbert. At the time of moving to Leicester, we stayed with him and Alma, at his farm, The Leys. He had many musical, technical and nautical interests that were always a pleasurable digression to discuss. So many characteristics of Herbert come through from every interaction I had with him, and I am very happy to have been able to pay this tribute. Hebert was, or course, very proud of his Scottish Heritage, and retained his musical accent that was so easy to listen to, carrying erudition and distinction in its tones. I do remember a meeting that ended with a Burns Night supper, where we had the privilege of hearing Herbert giving the most authentic recitation of the words of The Bard.

So I will end with some other words that are appropriate from Robbie Burns’ “Epitaph on my Own Friend”:

An honest man here lies at rest,
As e’er God with His image blest:
The friend of man, the friend of truth;
The friend of age, and guide of youth:
Few hearts like his, with virtue warm’d,
Few heads with knowledge so inform’d:
If there’s another world, he lives in bliss;
If there is none, he made the best of this.

Pat Heslop-Harrison.

Mine was one of four Tributes delivered at Professor Macgregor’s Thanksgiving Service. Others covered his early years including National Service, his Family, and life during his retirement in Topsham, Exeter.

 

Edits 14/8/18: hyperlinks, explain B&Q as shop.

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Complete mitogenomes from Kurdistani sheep – abundant centromeric nuclear copies representing diverse ancestors

Kurdistani sheep breeds and part of their mitochondrial genome From Mustafa et al. Mitochondrial DNA part A 2018.

Kurdistani sheep breeds and part of their mitochondrial genome From Mustafa et al. Mitochondrial DNA part A 2018.

341. Mustafa SI, Schwarzacher T, and Heslop-Harrison JS. 2018. Complete mitogenomes from Kurdistani sheep: abundant centromeric nuclear copies representing diverse ancestorsMitochondrial DNA Part A https://doi.org/10.1080/24701394.2018.1431226 (publisher – see free publisher link below if you can’t access) AND Mustafa et al_2018 AuthorVersion

The geographical center of domestication and species diversity for sheep (Ovis aries) lies around the Kurdistan region of Northern Iraq, within the ‘Fertile Crescent’. From whole genome sequence reads, we assembled the mitochondrial genomes (mtDNA or mitogenome) of five animals of the two main Kurdistani sheep breeds Hamdani and Karadi and found they fitted into known sheep haplogroups (or matrilineages), with some SNPs. Haplotyping 31 animals showed presence of the main Asian (hpgA) and European (hpgB) haplogroups, as well as the rarer Anatolian haplogroup hpgC. From the sequence reads, near-complete genomes of mitochondria from wild sheep species (or subspecies), and even many sequences similar to goat (Capra) mitochondria, could be extracted. Analysis suggested that these polymorphic reads were nuclear mitochondrial DNA segments (numts). In situ hybridization with seven regions of mitochondria chosen from across the whole genome showed strong hybridization to the centromeric regions of all autosomal sheep chromosomes, but not the Y. Centromeres of the three submetacentric pairs and the X chromosomes showed fewer copies of numts, with varying abundance of different mitochondrial regions. Some mitochondrial-nuclear transfer presumably occurred before species divergence within the genus, and there has been further introgression of sheep mitochondrial sequences more recently. This high abundance of nuclear mitochondrial sequences is not reflected in the whole nuclear genome assemblies, and the accumulation near major satellite sequences at centromeres was unexpected. Mitochondrial variants including SNPs, numts and heteroplasmy must be rigorously validated to interpret correctly mitochondrial phylogenies and SNPs.

Keywords: nuclear mitochondrial DNA segments (numts); mitogenome diversity; massively parallel sequencing; fluorescent in situ hybridization; sheep domestication

Free link to paper from publishers: http://www.tandfonline.com/eprint/YSxE33mSJiBMVEuMXIyv/full (limited to 50 uses so please look at author version first or use your academic library link via DOI below!)

341. Mustafa SI, Schwarzacher T, and Heslop-Harrison JS. 2018. Complete mitogenomes from Kurdistani sheep: abundant centromeric nuclear copies representing diverse ancestorsMitochondrial DNA Part A https://doi.org/10.1080/24701394.2018.1431226 () Mustafa et al_2018 AuthorVersion

 

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Morphology, adaptation and speciation

Plant adaptation arises from their morphology, itself a product of evolution and development. In this figure, the aspects and interactions of research at different levels are shown, with the work having implications across botany, including understanding plant phylogeny and speciation, and for ecology and ecosystems.

Plant adaptation arises from their morphology, itself a product of evolution and development. In this figure, the aspects and interactions of research at different levels are shown, with the work having implications across botany, including understanding plant phylogeny and speciation, and for ecology and ecosystems.

Heslop-Harrison JS. 2017. Morphology, adaptation and speciation. Annals of Botany 120(7): 621-624. https://doi.org/10.1093/aob/mcx130The study of plant evolution and development in a phylogenetic context has accelerated research advances in both areas over the last decade. The addition of a robust phylogeny for plant taxa based on DNA as well as morphology has given a strong context for this research. Genetics and genomics, including sequencing of many genes, and a better understanding of non-genetic, responsive changes, by plants have increased knowledge of how the different body forms of plants have arisen. Here, I overview the papers in this Special Issue of Annals of Botany on Morphological Adaptation, bringing together a range of papers that link phylogeny and morphology. These lead to models of development and functional adaptation across a range of plant systems, with implications for ecology and ecosystems, as well as development and evolution.

The study of evolution and development (evo-devo) has advanced plant research including speciation (Fernández-Mazuecos and Glover 2017); and as Theodore Dobzhansky stated in 1973, ‘nothing makes sense except in the light of evolution’. But what options are there for plant architecture to evolve – from the gene to cellular to organ and on to whole plant and ecosystem level? As suggested in the mind-map of Fig. 1, we are now in a good position to exploit data from morphological and genetic studies to understand the key processes of evolution and development, taking those results to find their impact on ecology and ecosystems. The papers in this special issue cover a diverse range of species, organs (related to leaves, roots and flowers) and approaches (from advanced microscopy to DNA fingerprinting), to show how modern plant studies can be integrated to lead to models of evolution and understand plant development in the broadest context.

 

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Crop Improvement – Plant Nuclear Genomes

328. Heslop-Harrison JS. 2017. Crop Improvement: Plant Nuclear Genomes. Encyclopedia of Applied Plant Sciences 2nd Edition. In proof.

Plant breeders work with large amounts of DNA sequence information including the sequences of all genes and the repetitive DNA that makes up the majority of most genomes. Knowledge of the diversity and evolution of the sequences is proving important to developing DNA markers, indentifying genetic and QTL characters, the selection of breeding lines and the measurement and exploitation of biodiversity. Nuclear DNA sequence information can be used for improvement all crop species with directed superdomestication approaches leading to improved performance, resistances to biotic and abiotic stress, and greater environmental sustainability to meet the challenges of increasing populations and climate change.

Keywords

Biodiversity, Breeding, Epigenetics, Genetics, Genomics, Genomes, Repetitive DNA, Traits, Plastomes, Chloroplasts, Markers, QTL, Chromosomes, Genome size

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Repetitive DNA in the catfish genome- rDNA, microsatellites, and Tc1-mariner transposon sequences in Imparfinis

Repetitive sequence organization on chromosomes of a Brazilian catfish species, important for understanding their evolution and biodiversity. Gouveia et al., Journal of Heredity, 2017.

Repetitive sequence organization on chromosomes of a Brazilian catfish species, important for understanding their evolution and biodiversity. Gouveia et al., Journal of Heredity, 2017.

339. Gouveia JG, Wolf IR, Vilas-Boas LA, Heslop-Harrison JS, Schwarzacher T, Dias AL. 2017. Repetitive DNA in the catfish genome: rDNA, microsatellites, and Tc1-mariner transposon sequences in Imparfinis species (Siluriformes, Heptapteridae). Journal of Heredity 108(6): 650-657.

Journal link: https://doi.org/10.1093/jhered/esx065 Author PHHGouveia_CatfishRepeatsAuthorVersion

Physical mapping of repetitive DNA families in the karyotypes of fish is important to understand the organization and evolution of different orders, families, genera, or species. Fish in the genus Imparfinis show diverse karyotypes with various diploid numbers and ribosomal DNA (rDNA) locations. Here we isolated and characterized Tc1-mariner nucleotide sequences from Imparfinis schubarti, and mapped their locations together with 18S rDNA, 5S rDNA, and microsatellite probes in Imparfinis borodini and I.  schubarti chromosomes. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in heterochromatin blocks with small accumulations in the terminal and interstitial regions of I. borodini and I. schubarti. Tc1/Mariner was coincident with rDNA chromosomes sites in both species, suggesting that this transposable element may have participated in the dispersion and evolution of these sequences in the fish genome. Our analysis suggests that different transposons and microsatellites have accumulated in the I. borodini and I.  schubarti genomes and that the distribution patterns of these elements may be related to karyotype evolution within Imparfinis.

Subject area: Genomics and gene mapping
Key words: genome evolution, karyotype evolution, MITEs, transposable elements

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