While the shamrock might have provided St. Patrick with a simple metaphor for the Christian Trinity, unraveling the complexities of its genetic variation is proving no easy task. But one group of Irish researchers has recently gotten lucky in describing the genetic variation in Trifolium repens, also known as the white clover.
Milbourne’s group isn’t the first to look at the clover genome: the white clover was first sequenced in 2007 (2), and the genetic region believed to hold the key to the four-leaf variant of the plant was identified in 2010 (3). White clover has four sets of chromosomes, uses separate male and female parts to breed, and prefers not to inbreed. Overall, the clover genome contains many alleles—alternative forms of the same genes—which makes unraveling its genetics particularly challenging.
In their recent study, Milbourne’s group identified nearly 210,000 single DNA base-changes, or SNPs, in the clover genome to determine whether the variation originated in the plant’s extra sets of chromosomes or from its alleles.
To do that, Milbourne’s team sequenced samples from eighth-generation, inbred white clover lines. The key to the experiment was to have lines in which the two individual, ancestral sub-genomes of the clover were completely homozygous, meaning they had the same DNA sequences from the parent plants for a gene and related trait. It took years of inbreeding and selection to get the lines right and even then concerns about the extent of homozygosity in the inbred lines remained.
The team then started sequencing, first with 454-pyrosequencing to generate about 760,000 transcripts from the clover samples and then with Illumina next-generation sequencing on three other white clover samples to generate 14 million transcript reads from a mixed sample of 24 divergent white clover genotypes and 50 million reads on 2 additonal eighth generation white clover inbred lines.
Finally, the team mapped the results onto the reference transcript set, creating a SNP library for white clover. Then they determined the source of the SNPs, whether they were from the plant’s alleles or from its extra chromosomes sets.
While previous groups had worked out some of this variation (4), Milbourne said that next-generation sequencing allowed them to get a much more detailed view. "We freed ourselves from the constraint of having to PCR amplify individual genes and sequence them one by one, allowing us to scale up to tens of thousands of sequences," Milbourne said.
But the method could not identify which ancestral subgenome of white clover the variations originated from. In addition, it did not explain why some of the extra chromosome pairs are as similar to each other as allelic variants from the same sub-genome would be. Unraveling these complexities will keep his lab busy going forward.
The white clover is a good example of the gulf that exists between the ease with which scientists can study the genomes of model species such as Arabidopsis and the challenges of working with real crop plants such as white clover, said Milbourne. “It’s really a very exciting time for white clover genomics,” he added.
- Nagy, I. et. al. (2013). “A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover.” BMC Genomics. 14(100): 1-36.
- Zhang, K. et. al. (2007). “Genome mapping of white clover (Trifolium repens L.) and comparative analysis within the Trifolieae using cross-species SSR markers.” Theor Appl Genet. 114(8): 1367–1378.
- Tashiro, R. et. al. (2010). “Leaf Trait Coloration in White Clover and Molecular Mapping of the Red Midrib and Leaflet Number Traits.” Crop Science. 50 (4): 1260-1268.
- 4. Sawbridge, T. et. al. (2003). "Generation and analysis of expressed sequence tags in white clover (Trifolium repens L.)". Plant Sci 2003, 165:1077–1087