Soybean cyst nematode (SCN) is a major pest of soybean which causes devastating losses to soybean industry every year. SCN management practices rely on the resistant genetic source, majorly the rhg1 and Rhg4 loci. Our goal is to alter rhg1 and Rhg4 alleles in the elite susceptible cultivar through prime editing and enhance its resistance to SCN. CRISPR Prime editing is recently (2019) invented tool also called as search and replace tool, which alters DNA (SNPs/haplotypes) in the host genome without leaving any foreign DNA. This tool has been successfully evaluated and used in the mammalian field. Its application in plants has not been shown so far. Here, we are employing prime editing to alter novel haplotypes in susceptible soybean lines. We cloned prime editing components such as dead-Cas9 (dCas9) fusion with reverse transcriptase gene and prime editing guide RNAs of Rhg4 locus haplotypes. Currently, we are evaluating the prime editing components in Soybean hairy roots. Additionally, we are developing novel germplasm populations combining new sources of SCN resistance genes (independent of rhg1 & Rhg4) into elite breeding lines for durable SCN resistance. Haplotype and structural variation analysis demonstrates that, these new SCN resistant lines shows unique and novel haplotypes compared to other existing SCN resistant soybean lines.
Additionally, we are studying the landscape of soybean epigenome for SCN resistance to better understand the epigenetic control of SCN resistance and decipher the methylated regions and/or cis elements at the promoter region of the Rhg4 and rhg1 genes that leads to SCN resistance in soybeans.
Through TILLING-by-Seq, we have developed six mutants to access new genes for SCN resistances. The SCN phenotyping of these mutants showed two of them have higher cyst count compared to parent genotype indicating involvement of these mutations in SCN resistant mechanism.