Genome Resequencing for Mutant Identification

As most biologists will be aware, the cost of DNA sequencing has been falling well in advance of the costs predicted by Moores law (although argued by Neil Hall a few years ago, this might not have been the best thing to happen, intellectually at least).

Instead of simply sequencing many genomes for the sake of it, this also offers opportunities for researchers to use this technology to ‘do-science’ that might previously have been prohibitively laborious or expensive. One such area where this is true is in the identification of novel mutations in plants, especially in Arabidopsis.

Classic approaches to identity the location of an EMS mutation involved mutant identification, backcrossing, selection, rough mapping by PCR or CAPS markers, probably more crossing and then a little guesswork toward the end..…..before using Sanger sequencing to identify what you hope is the causative mutation. Even with a strong following wind this process could take upwards of a year……. many a 1990s PhD thesis was written off the back of mutant identification. In contrast it is now relatively cheap to resequence the Arabidopsis genome so a lot of time can be taken out of this process. In addition, resequencing can remove some of the difficulty involved with selective of mutants that have a subtle phenotypes wherein inaccurate selection of putative mutants would significantly set back the process.

Back in 20111, Anthony Hall’s group in Liverpool University used resequencing in parallel with classic genetics to identify the lesion in the novel early bird1 gene (ebi1), which has a defect in function of the circadian clock. In this case ebi1, which was generated using EMS, was backcrossed 4 times to reduce the number of EMS-induced SNPs not associated with phenotype, and then sequenced alongside the original wildtype plant (from the WS ecotype). The critical part of the protocol came in the power of the software they used to detect homozygous SNPs in the ebi1 line. Indeed the researchers ran into some difficulties due to a high number of SNPs they initially identified. However, when they combined altering the stringency of SNP-calling together with classical rough mapping they were left with approximately 30 SNPs to finally assess. Using a priori knowledge of proposed gene function and by investigating expression changes in these candidates they ultimately identified a novel mutant. Although this process was ultimately successful, it took some extra time due to the difficulty of mutant selection, optimization of the SNP-calling software and subsequent analysis of gene expression.

A recent paper from the lab of Lucia Strader at Washington University in St Louis shows how powerful resequencing can be if you are using a robust method of mutant selection. In their case they isolated mutants with a defect in the root growth response to ABA, which is an unequivocal phenotype to score. They backcrossed their initial mutants, selected for ABA resistance in F2 generation before resequencing these resistant plants. Using this process the authors report that they narrowed their search to between 3-10 candidate genes and that they have subsequently identified novel (unpublished) genes using this method. In addition, as an exemplar of their protocol they used it to isolate novel alleles of known ABA-resistant mutants.

Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167
Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In parallel they used a similar protocol to the Hall lab where they resequenced non-backcrossed plants and then selected SNPs that only lay within exons.Using this approach they identified between 100-200 homozygous SNPs, a potentially fifty-fold increase compared to their other method. Therefore when you are working with a strong robust phenotype it is probably worth the extra time to obtain a back-crossed population in order to have greater confidence you are isolated your mutant of interest.

The authors importantly note that one limitation of this protocol is that by only selecting for exonic mutations, they are removing the possibility of identifying mutants with splicing or non-coding defects, which may in turn rule out a number of candidate genes.

 

For me the take-home message from this second study is that if you have a robust phenotype to select for and are confident that your mutation is novel then use of ever-improving NGS is now a time and cost effective way of mutant identification.

In fact this technology might inspire a return to the forward genetic screens of the 80s and 90s , with the aim of identifying novel genes involved in well characterised signaling pathways……..except that PhD students might now have to characterise 10 novel genes prior to graduation….

CoGe at PAGXXIII

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Published on: January 11, 2015

Happy New Year! We at GARNet enjoyed a long Christmas break and some of us have returned to work via California! Ruth, Jim and I are in San Diego this week for the Plant and Animal Genome Conference (PAG).

PAG is an enormous conference – take a look at the Twitter stream (PAGXXIII) for an idea of how many sessions run at any one time. Yesterday I went to sessions on Ontologies, Brassica and Tritaceae, and I thought I’d quickly update our blog readers about a workshop about the CoGe online tool. I mentioned CoGe in this post about the EPIC conference and it’s also featured in the June 2013 edition of GARNish.

Eric Lyons, one of the creators of CoGe, began the session by explaining that CoGe is a platform for managing, visualising, analysing and comparing genomes. It can deal with unlimited numbers of genomes of unlimited size—though there is a limit for the number of annotations per genome—and while there are tools set up for ease of use, users can perform custom, on-the-fly analysis too.

Throughout the session, Lyons was clear that CoGe is ‘Powered by iPlant.’ It uses iPlant middleware to enable data storage, universal log-in and much more functionality that the user might not be aware of but which makes their experience smooth and relatively stress-free. (more…)

What makes one species different from another?

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Published on: December 10, 2013

In the third of our series of blog posts Celebrating Basic Plant Science, Catherine Kidner from Edinburgh’s Royal Botanic Garden explains how she pinpoints what makes a species a species. This sheds light onto what drove that species’ evolution. Catherine spoke about her research at a conference earlier in the year – you can see her talk in this video

Catherine Kidner uses genomic data to understand why Begonia like these have evolved into different species

There are likely to be about 9 million species on Earth (not counting bacteria). Each species has traits which define it and allow it to thrive in its niche. My research group at the Royal Botanic Garden in Edinburgh is trying to understand the diversity around us, so we need to know what these traits are and how they contribute to the success, or otherwise, of the species.

There are difficulties with this approach to understanding diversity. For a start, traits we think are important may not be that important to the species concerned. Also, some traits critical to a species’ success might be difficult to measure, for example phosphate uptake by roots; or be seen only on very specific occasions, like response to a particular pathogen.

Using genetics to define differences between species

We get around these problems by looking at genetic differences between species. New sequencing technologies make it relatively quick and easy to sequence the genome of an individual. 

In a typical genome, around 25 000-50 000 genes code for proteins. If we want to know how two species differ we can compare sequences of these protein coding genes and see which types of gene differ the most between species. These changes in gene sequences mean the proteins work differently.

Not all genes are expressed, that is translated into proteins, all the time, and we can see which sets of genes are expressed at particular times and in different organs, like petal or leaf. So when looking at how species differ, we can also look at which genes show the biggest changes in expression level, which would mean one species having more of a particular protein than the other.

Having a list of which genes show sequence changes and which show expression changes is not much help if it’s just a list of genes xzyabc and rst. What really makes this a useful technique are the huge databases which have been built up over the past 20 years. Work in model species such as yeast, Arabidopsis, mouse and Drosophila have determined functions for many genes in typical genomes. We can match the genes in our ‘interesting genes’ lists to sequences from these model organisms to find out that, for example, gene xzy looks like a disease resistance gene, or gene abc looks like a gene that controls root growth rates.

A typical comparative study might highlight hundreds of genes which differ between species, so even with good descriptions of function we still have a lot of data to sift though to find patterns. We can simplify the lists by using GO (Gene Ontology) terms. This is a way of describing what genes do in a very defined way. 

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Staying together: green beginnings

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Published on: November 5, 2013
This seaweed, Ulva linza, would only exist as undifferentiated cells if not for bacterial signals

The second of our series of blog posts Celebrating Basic Plant Science is written by Juliet Coates, Lecturer in Molecular Genetics at the University of Birmingham.

Living organisms can be categorised in a number of ways, but one very obvious “either/or” distinction is between organisms that are made up of a single cell – unicellular organisms – and those that are many-celled, or multicellular.

The multicellular state arose many times during evolution: animals, plants, algae, amoebae, fungi and bacteria can all be multicellular. Multicellular organisms completely underpin life on Earth as we know it today – and they all must have evolved from single-celled ancestors. We understand a little of why they might have done so, as being multicellular gives a number of competitive advantages: increased size and improved nutrient collection being just two. Yet how multicellular organisms came to be is a key biological problem that is still largely unanswered.

I am a plant scientist, so I am particularly interested in the origins of multicellular green things: plants and algae. Without becoming multicellular, plants would never have colonised the land, and the evolution of multicellular plants and algae was key in shaping our climate, our ecosystems and our oxygen-rich atmosphere. How green multicellularity arose seems to me to be a really fundamental thing to understand, but it is a little-addressed question. Here I’ll give an overview of the important findings to date about the evolution of multicellularity.

 

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Plant science – making an impact on scientific publishing

Categories: Arabidopsis, resource
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Published on: September 5, 2013

This year is proving to be a good year for plant science publications. So far there have been special plant science issues in Science and Genome Biology (and I have it on good authority that there will be plant synthetic biology special issue of another journal coming soon) as well as a landmark birthday for New Phytologist.

Special Issues for Plant Science

The open access journal Genome Biology published their Plant Science Special Issue in June 2013. It was guest edited by Mario Caccamo, acting director and Head of Bioinformatics at The Genome Analysis Centre. He discusses the issue and explains the importance of plant genomics, alongside Dale Sanders and other experts, in this podcast from Biome, BMC’s online magazine. The special issue itself features a whole host of UK researchers, including  Cristobal UauySebastian SchornackAnna Amtmann and Edgar Huitema.

The Science Special Issue, published just last month, unsurprisingly had a much broader focus – Smarter Pest Control. The featured reports take a global look at issues surrounding crop protection from pests, including RNAi-based pesticides, possible health problems caused by traditional pesticides, and tracking the effects of pesticides in wild animal populations.

New Phytologist Celebration

The Lancaster based journal New Phytologist, founded in 1902, is celebrating 200 volumes in October. By my reckoning, it’s the second oldest plant science journal in the world, after Annals of Botany which began life in 1887 as the Journal of Botanical Science (special mention for strictly botany journal, Flora). There is an incredible celebratory Virtual Special Issue of New Phytologist available here, featuring historic articles from throughout the journal’s lifetime including a 1904 critique of the then fashionable field of plant-based ecology from the great man himself, Sir Arthur Tansley.

Arabidopsis UK research roundup

On a related more local note, our new team member Lisa has been searching the literature each week for publications from UK Arabidopsis or other basic plant science researchers. She’s posting the Arabidopsis Research Round-up to the GARNet News pages, so check it out if you want to keep up with new research from your UK colleagues. If you’ve been published and want to make sure we spot your paper (we’re not perfect!), feel free to email Lisa at lisa@garnetcommunity.org.uk to let her know.

Were you there? Arabidopsis as a model plant

Categories: Arabidopsis
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Published on: February 26, 2013
A reproduction of a painting by C. A. M. Lindman (1856–1928)

The history of Arabidopsis as a model organism and of the Arabidopsis Genome Project is the story of the birth of modern plant science, full of visionaries and phenomenal teamwork. No doubt some readers of this blog were present at the dawn of the Age of Arabidopsis, but many others think of Arabidopsis as an established part of the plant science landscape. I myself can’t imagine pre-Arabidopsis plant research, before seed stock catalogues, genome sequences, and BLAST. For all you early career Arabidopsis researchers  this is your opportunity to catch up on the history of this most-researched weed. For those of you who remember it, I hope I got most of it right!

German scientist Friedrich Laibach first suggested Arabidopsis as a plant model species in 1943. He noted that A. thaliana was easy and fast to grow, showed a lot of natural variation, was amenable to cross-breeding between varieties, and generated a lot of progeny. By the 1960s a number of researchers in Germany and a few elsewhere were working on Arabidopsis.

The first annual Arabidopsis Information Service was put together by Gerhard Röbbelen in 1964, and you can read an electronic version of it on TAIR. It is interesting to see the kind of research going on in the ’60s – authors report X-ray and biochemical mutants, growth and development under certain conditions, and methods to induce mutations and grow sterile seedlings on agar. A year later, the first Arabidopsis Symposium met in Göttingen. You can see a picture of the delegates here.

It took twenty more years for Arabidopsis to became widely used worldwide. Albert Kranz, at the Botanical Institute of the Johann Wolfgang Goethe University, collected and maintained Arabidopsis seed stock for the community and took over the Arabidopsis Information Service in 1974. During this time, early identification of embryo-lethal mutants and the small genome size, and by the end of the 1980s, reliable Agrobacterium-mediated gene transfer, were all added to the list of benefits to working with Arabidopsis.

In 1989, James Watson, by then the Director of Cold Spring Harbour Laboratory, called a meeting to discuss the use of Arabidopsis as a model for genetic research. A year later, the newly formed Multinational Arabidopsis Steering Committee (Marc van Montagu, Caroline Dean, Richard Flavell, Howard Goodman, Maarten Koornneef, Elliot Meyerowitz, Jim Peacock, Yoshiro Shimura and Chris Somerville) published a report outlining plans to sequence the whole Arabidopsis thaliana genome. At the time, the project sounded overly ambitious and unlikely to be completed – A. thaliana has a relatively small genome, but at around 120 million base pairs long it was a mammoth project. (more…)

New Methods and Resources (I)

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Published on: February 5, 2013

On this blog, I highlight a new method or resource pretty regularly. I used to work in what I think is a fairly normal UK plant science lab, so I try to comment on aspects I would have found useful to know about, for example if the method requires a machine not every lab has, or if it is unclear about anything. However, there are many, probably excellent, new open software and techniques which I don’t highlight on the blog because I am completely unfamiliar with their background.

For today, here’s the first part of a round-up of plant methods and resources published over the last few months. If you have used them, feel free to let me know how they worked in the comments, or through email or Twitter. And if you would like to review a method or resource for this blog, please get in touch!

iRootHair is a free, online, curated, expandable database of root hair genomics. Kwasniewski et al. (2013; Plant Phys. 161:28-35) built the database, which currently includes information about 153 root-hair related genes. The majority of the genes are from Arabidopsis, but maize, rice, tomato, and barley genes are also included. There is a page showing figures of various root phenotypes, which users can click through to see the genes associated with a specific phenotype; and a similar one for root processes like tip growth. (more…)

Plant Science for Christmas

Categories: something fun
Comments: 1 Comment
Published on: December 20, 2012

Plants make Christmas, from the wreath on the door to the brussels sprouts on the table. In celebration of plant science and this most planty time of year, here’s some Christmassy plant science for you to enthral (or bore?) your nearest and dearest with next week. Perhaps while some of them are trying to watch the Made in Chelsea Christmas Special…

The Holly and the Ivy: Holly reacts to herbivores by making some leaves prickly while leaving others smooth – a form of heterophylly, where a plant has two or more types of leaf. This story from Science Daily also features ivy – science inspired by S’Cliff Richard himself!

I Saw Mummy Kissing Santa Claus: Mistletoe is already a ‘complementary and alternative’ cancer treatment (see the National Cancer Institute for more information), but a new study indicates it may become the source of a new mainstream anti-cancer drug. Researchers from the University of Adelaide have shown that an extract from mistletoe species Fraxini effectively reduces the viability of colon cancer cells, and is more potent than a chemotherapy drug.

We Three Kings: Frankincense is harvested from Boswellia papyrifera by ‘tapping’ the tree trunk and collecting the resin. Over-harvesting a tree ironically causes resin production to fall or cease as the tree expends resources on healing the wounds caused by tapping, and can even kill the tree as pathogens take advantage of the damage to the trunk. The Annals of Botany blog highlighted a paper published in Annals of Botany about the anatomy of the resin secretory system, and how the knowledge can improve sustainability of frankincense harvesting.

Oh Christmas Tree: If you have a live Christmas tree, it’s likely to be very similar to the conifers that dinosaurs roamed around. A study published in BMC Biology in October (Pavy et al., 2012) showed that the genomes of spruce and pine, which diverged 100 million years ago, have high synteny and co-linearity, suggesting no major genome changes have occurred. Senior author on the paper, Professor Jean Bousquet from Université Laval in Quebec, said, “Conifers appear to have achieved a balance with their environment very early. Still today these plants thrive over much of the globe. In contrast, flowering plants are under intense evolutionary pressure as they battle for survival and reproduction.”

Finally, for non-planty but very funny Christmas-based ‘science’ (inverted commas necessary), check out Dr Molecule’s latest blog post.

Image credit: Holly (ilex aquifolium) by Alfred Borchard; Pine Wood by Hajnalka Ardai Mrs., all via stock.xchng.

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