Identification and characterization of new low germination stimulant 1 (lgs1) sorghum
We sought to determine if there were Striga resistant genotypes in the SAP. The best characterized genetic cause of Strigaresistance is the LOW GERMINATION LOCI 1 (LGS1 ) that leads to the low germination stimulant phenotype. The causative gene (Sobic.0011G112600) encodes a sulfotransferase whose loss of function causes a shift from high to low potent SLs (Gobena et al. 2017). Sorghum genotypes with a deletion in the LGS1 region are poor inducers of germination and are Striga resistant.
To determine if any of the SAP genotypes had a deletion on theLGS1 region, we performed a polymerase chain reaction (PCR) to amplify the sulfotransferase gene. A positive PCR is indicative of an intact gene and a band absence represents a gene deletion. To ensure that the lack of a band is not due to PCR failure, our PCR was designed to simultaneously amplify the sulfotransferase and the housekeeping gene, ubiquitin. Analysis of 373 genotypes, using LGS1/lgs1 maker identified twelve genotypes that had a no amplification product for the sulfotransferase gene. Wild type (LGS1 ) sorghum showed two amplicons, Ubiquitin (223 bp) and sulfotransferase (628 bp) signifying an intact gene consistent with the Striga susceptible Shanqui Red, used in this study as a susceptible check while lgs1 showed a single band at 223bp resulting from Ubiquitin amplification but not sulfotransferase (Figure 2a). Absence of the 628 band is consistent with a deletion in the LGS1 loci previously described in the Strigaresistant SRN39 used in this study as the resistant check. In total, we identified 12 lgs1 genotypes from the screened 373 SAP. Two of these genotypes (PI 656040) and (PI 533976) had been previously described in Bellis et al. 2020.
The hallmark of the lgs1 mutations is the ability to direct the SL biosynthetic pathway towards orobanchol instead of 5-deoxystrigol. We therefore sought to determine if the observed Striga resistance in the SAP lgs1 genotypes was associated with production of orobanchol. LC-MS-MS analysis of SLs from the root exudates of SAPlgs1 revealed a predominant peak that corresponded to orobanchol (RT=10.86 mins) while SAP LGS1 lines showed a peak identical with that of 5-deoxystrigol (RT =14.59 mins) (Figure 2b). Consistently, mutant SAPlgs1 genotypes alongside the resistant check SRN 39 had high levels of orobanchol (Figure 2d) while wild type SAP LGS1 and Shanqui Red, a well characterized LGS1 genotype, predominantly produced 5-deoxystrigol (Figure 2d). These findings affirmed that mutations in LGS1 lead to changes in the types and amounts of SL emitted and that Striga resistance is associated with production of orobanchol as the major SL in sorghum.