CRISPR IDENTIFIES SHATTERING GENE IN A WILD SPECIES, Setaria viridis
DR. SUJAN MAMIDI
HudsonAlpha Institute for Biotechnology
Photorespiration is a respiratory process in higher plants, where oxygen is taken in and gives out carbon dioxide, opposite to what photosynthesis does. This is supposed to decrease yield in crops to about 20-30%. Most plants are C3 (carbon dioxide fixation takes place only at one place), and don’t have photosynthetic adaptations to reduce photorespiration. In C4 plants, it occurs twice (first in mesophyll cells and second in bundle sheath cells). C4 photosynthesis leads to higher productivity in several major food crops and bioenergy grasses, including maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), millets (e.g. Panicum miliaceum, Pennisetum glaucum, and Setaria italica), Miscanthus giganteus, and switchgrass (Panicum virgatum). Gains in productivity are associated with C4 photosynthesis and also have improved water and nitrogen use efficiencies.
C4 photosynthesis is most productive under the hot, dry conditions that are predicted to become more prevalent with climate change. Engineering C4 traits into C3 crops is an important target for crop improvement. Setaria viridis (green millet) is a close relative of several major feed, fuel, and bioenergy grasses and serve as a model crop to understand C4 physiology better. It is a diploid, has a small genome, has short stature, requires simple growth requirements, has short life cycle (seed to seed in 8–10 weeks), are self-compatible, and a single inflorescence can produce hundreds of seeds. In addition, transformation is efficient and amenable to CRISPR–Cas9-mediated mutagenesis.
As in most wild species, seeds of S. viridis fall off the plant at maturity, a process known as shattering. This is essential for dispersal in natural ecosystems, but undesirable for cultivation. Domestication in many cereals is often associated with selection of non-shattering plants. More often, identifying such loci was based on crosses between domesticated plants and their wild progenitors, and identifying quantitative trait loci (QTLs).
We identified a gene called Less Shattering 1 (SvLes1) using association mapping1. To validate SvLes1 as the causal gene, we used CRISPR–Cas9 to create additional alleles. We disrupted the wild-type, high-shattering allele SvLes1-1 to create several nonfunctional alleles. Sequence analysis revealed an adenine insertion at position 149 of the transcript, leading to a frameshift mutation which is predicted to completely abolish gene function, thereby creating non-shattering plants.
In the domesticated Setaria italica (foxtail millet), we discovered a ~6.5-kb copia transposable element (copia38) inserted between the two Myb domains and creates a loss-of-function allele, which produces a low-shattering phenotype. This is the first time we cloned such loci via association studies of natural diversity, that too in a wild population. We also identified a gene (SvLiguless2) associated with leaf angle, which determines how much sunlight leaves can get and in turn serves as a predictor of yield.
The final version 2.0 release is a complete telomere-to-telomere chromosomal assembly, containing 395.1 Mb of sequence in 75 contigs with a contig N50 of 11.2 Mb. The genome has 38,334 gene models and 14,125 alternative transcripts. In addition, we sequenced 598 diverse lines (mean of 56 million high-quality paired-end reads) and each library was subsequently assembled into a pan-genome. Pan-genome has about 51,000 genes, which represents all the genes that are present in a species. Building the pangenome allows us to identify association between presence/absence variation and phenotypes. Within US, four subpopulations were identified that are similar to worldwide collection. This suggests that this crop was introduced into US through multiple routes from Eurasia. Within US, we associated climatic variables and identified candidate genes associated with climatic adaptation. This climate associated markers/genes can help us identify/develop new varieties that would be necessary for adapting to changing climates.