Genetic Analyses in Pearl Millet
Pearl millet is an important food crop in India and Sub-Saharan Africa, and is grown for forage in the US and Australia. Most pearl millet accessions cultivated in India, the US and Australia are semi-dwarfs due to the presence of a single recessive gene, d2. Reduced plant height increases yields under high fertilizer inputs due to reduced lodging. The higher leaf to stem ratio in the semi-dwarf accession also provides better forage quality. Despite the economic importance of the pearl millet dwarfing gene, its identity is unknown. Our project aims to isolate and characterize the gene underlying d2 using a map-based cloning approach. Because genomic sequence for pearl millet is not yet available, we will heavily draw on comparative information from the related grass species. Upon isolation, gene identity will be demonstrated through transformation. Gene charactization will include identification of the mutation that causes the d2 phenotype, and detailed expression analyses.
Abcb1, a gene encoding an ATP-binding protein that plays a role in polar auxin transport, was identified as the likely candidate underlying d2.
See Parvathaneni et al. (2013) Genes, Genomes and Genetics 3: 563-572 for more information
The structure of abcb1, the gene underlying the semi-dwarf d2 phenotype in pearl millet, is variable across the grasses with multiple introns having undergone recurrent loss. Only intron 7 is present in all grasses analyzed and this intron carries an 8-bp motif that is highly conserved across monocots and dicots indicating functional importance. Transformation of intron deletion constructs in the Arabidopsis abcb1/abcb19 double mutant will indicate whether intron loss in the abcb1 gene affects gene expression patterns.
We are interested in unraveling the mechanism(s) behind intron loss. We hypothesize that the efficiency of intron removal by the spliciosome is correlated with the probability of intron loss. Hao Wang and colleagues (Wang et al. 2014) have identified ~100 genes in grasses that contain introns that have been lost recurrently. This data set is perfect to test this hypothesis. Nascent pre-mRNA, poly(A) mRNA from nuclei and mature mRNA from Arabidopsis and sorghum will be sequenced and used to perform a global analysis of the relative rates of intron removal by the spliceosome and the factors (e.g.intron length, intron position, sequence context around splice sites, alternative splicing) affecting these rates. Analysis of three types of RNA will reveal whether retained introns are spliced out post-transcriptionally and/or transported to the cytoplasm. Statistical analyses will be carried out to determine whether relative splicing rates are different in Arabidopsis and sorghum, and whether they are correlated with patterns of intron loss.
Katrien M. Devos (PI) - Institute of Plant Breeding, Genetics and Genomics, and Dept. of Plant Biology, University of Georgia, Athens, USA