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.