Single-nuclei genome sequencing: a breakthrough in studying Mycorrhizal fungi
By Kirsty Jackson (@kjjscience)
The Human Genome Project officially started in 1990 and the final polished version was released in 2006. The project cost billions of U.S Dollars and had scientists around the world working upon it. Since the announcement of the complete “rough draft” human genome in 2000 the genomes of many organisms have been sequenced and the cost of sequencing has reduced drastically. The phrase “Just sequence it” can now be found in many biology labs, said in the same blasé tone one would use when deciding what to have for dinner.
However, for many biological organisms genome sequencing is still quite a challenge. To put things into context, the nucleus of a human cell holds 23 pairs of chromosomes and in every cell in the human body those chromosomes are the same. Therefore if you took a sample of tissue containing 10 cells, you would have 10 times as much of exactly the same DNA. Humans are diploid (2n), therefore if you picked a chromosome (say 3), the information encoded the chromosome 3 you got from your Mum will not exactly match the chromosome 3 you got from your Dad. However, this difference is the same in every cell. So if you looked at multiple cells from the same person, the difference would become clear. In haploid organisms (1n), such as the model filamentous fungus Neurospora crassa, there is only one copy of every chromosome (per nucleus) which makes the genome puzzle even easier to put together.
To get a genome sequence, short sections of the genome are read and then these small fragments are matched together based on their overlapping sequences by a very powerful computer. This is why we say a genome is “assembled”.
There are some other types of genetic arrangements that are more difficult to sequence. Many plants can contain multiple different copies of a genome, such as common bread wheat, which is hexaploid (6n, or 3 pairs of chromosomes). In some organisms, such as the mycorrhizal fungus Rhizophagus irregularis, each nucleus can be different. In R. irregularis each fungal spore can contain many nuclei, and each nucleus will have different information inside them. If you can imagine, it would be like taking one nucleus from each person along your street and putting them all into the same cell. It is this multinucleate life-style that has made acquiring the genome of R. irregularis difficult. But there might be a light at the end of that genome tunnel.
A recent paper in PLOS Genetics describes how Lin et al. are trying to work out the genome of R. irregularis using new technologies and increased sequencing power. The authors stained the nuclei of spores green and then used a very tiny pipette (similar to the kind seen in IVF-treatment news reports) to select and retain individual nuclei (Figure 2).
They then amplified the DNA of each individual nucleus so that the quantity was high enough to read and assembled the genomes of 4 individual nuclei. The sizes of the genome of each nuclei was different (115 Mb, 90Mb, 71Mb and 95Mb) but when compared to each other, large sections were identical between nuclei. Therefore, although each nucleus is different, there is a core set of genes in all the nuclei. They also compared the individual nuclei to DNA obtained from sections of mycelium (containing many nuclei) and saw that the results were not much different.
Using gene prediction programs, Lin et al were able to predict 14073 genes are encoded by the genome. They were then able to compare these genes with other known fungi and found that ~9.7% of the genes had not been found in the genomes of other “sequenced” organisms and could be specific to mycorrhizal fungi more generally. There were also a surprising number of transposable elements in the genome, which could account for the varying sizes of genome between nuclei.
Currently, research on the symbiosis between R. irregularis and plants is heavily biased towards the plant. This new genomic information will enable scientists to study the fungal side in a lot more detail. It will also enable comparisons between the friendly mycorrhizal fungi and their disease-causing relatives, allowing a greater understanding of the difference and similarities between friends and foe.
About the author: Kirsty Jackson is a PhD student at the John Innes Centre, Norwich. She is studying bacterial and fungal symbioses with legumes and loves all things fungi! When she isn’t in the lab she is involved with organising science outreach events. Follow her on twitter (@kjjscience)
Lin K, Limpens E, Zhang Z, Ivanov S, Saunders DGO, et al. (2014) Single Nucleus Genome Sequencing Reveals High Similarity among Nuclei of an Endomycorrhizal Fungus. PLoS Genet 10(1): e1004078. doi:10.1371/journal.pgen.1004078