On creating a collection of deletion mutants in microRNA genes from sweet sorghum

- Essay in plant genetics & genomics -

Elucidating the genotype-to-phenotype relationship is essential for a comprehensive understanding of plant development and consequently, manipulation of traits important for agricultural productivity. Genetic resources for functional genomics in sorghum are lagging behind those developed for rice and maize. This constraint can be alleviated by developing a resource for reverse genetics in sweet sorghum in which microRNAs and their role in the regulation of plant traits relevant to the bioenergy industry can be addressed.

Concisely, it will help to create a collection of deletion mutants in sweet sorghum where null mutations can be identified by screening genomic deletions that span microRNA genes.

Although EMS mutagenesis has been widely used in both forward and reverse genetic screens, it has not been very successful in yielding point mutations in genes with lengths less than 1 Kbp. This is a significant limitation for the creation of point mutants in microRNAs, which are usually encoded by small transcriptional units. Most importantly, since many microRNA genes are arranged in clusters in the sorghum genome, the identification of a deletion big enough that can remove several microRNA genes copies at once would be greatly advantageous.

It is exciting to think on the screening of deletion mutations in microRNA genes and the characterization of their associated mutant phenotypes in terms of sugar content in stems, biomass production, and stress tolerance (drought and salinity); so that sweet sorghum's potential as energy crop can be improved through mutation breeding.

One possible approach would entail the irradiation of sweet sorghum seeds with fast neutrons as the mutagenic agent. The screening of deletions could then be performed through a combination of array hybridization and high throughput sequencing. The advantage of array hybridization is that a custom Affymetrix array can be constructed with probe sets that recognize the sequence of all annotated microRNA precursors in the sorghum genome together with their flanking sequences (2 Kbp of upstream and downstream sequence, respectively). The screening of deletion mutations across the genome can be performed through the hybridization onto the array with DNA from pools of M2 plants and from wild type sweet sorghum respectively. Deletions in any microRNA gene printed on the array can be detected at once by differences in DNA hybridization intensities between mutant and wild type samples.

Here, it is assumed that the genome sequence of the sweet sorghum cultivar used in the mutagenesis experiment is already available (and compared with the reference sequence of grain sorghum BTx623).

What it is really appealing about creating deletion mutants in sweet sorghum is that they can be subsequently transformed with allelic series of microRNAs from other sweet sorghum cultivars/varieties and test the effect of DNA polymorphisms on the phenotype. Furthermore, it will be necessary to perform sequencing of full length transcript in the desired sweet sorghum variety in order to characterize the architecture of microRNAs genes and compare them to the reference genome sequence.

The use of sweet sorghum as model system in the creation of additional genetic resources has a tremendous impact on the study of sugarcane, a related grass, because of the ploidy level in sugarcane it will be challenging to obtain phenotypes associated with deletion in microRNA genes since many other copies would still remain functional in the genome.