Molecular markers for defense response genes in sorghum

Abstract

Oligonucleotide primers made complementary to conserved sequences in phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chitinase (CHT), glucanase (GLU), cinnamyl-alcohol dehydrogenase dehydrogenase (CAD), 0-methytransferase (OMT), 3-hydroxy-3methylglutaryl CoA reductase (HMGR), glutamine synthetase (GS) and dihydrofolate reductase (DFR) genes previously cloned from other species were used to amplify segments of the equivalent genes from sorghum. From amplified fragments inserted into pUC18 in E coli, three putative CHS fragments, two CHTS, three GSs, one HMGR, one CAD, two OMTs and four PALs were obtained. Greater than 70% base sequence identity with homologous genes confirmed (PALI-1) and a genes confirmed that a 412bp clone (CAD13), a 535bp clone (PALI-1) and a 620bp clone (CHS2G) were derived from the coding regions of CAD, PAL and CHS, respectively. The other clones show <30% homology so most likely they did not originate from functional copies of the corresponding sorghum genes. Southern blots showed that CAD13, PALI-I and CHS2G each detected several EcoRI fragments. One fragment for each of the PALI-I and CHS2G probes was polymorphic in the parents of a mapping population, permitting a locus for each gene to be located on a sorghum RFLP linkage map. The three clones detected RFLPs among races of 53 accessions, but variation was rarely seen within a race. Northern blots showed that low levels of PAL and CHS MRNA were present constitutively. Messages from both genes accumulated more rapidly and to higher levels after inoculation with Bipolaris maydis than in uninoculated controls. No significant increase of either PAL or CHS MRNA was seen in seedlings of BTx635 and RTx7O78 after inoculation with Sporisorium reilianum. Differences in the timing and level of MRNA accumulation were detected in seedlings after inoculation with Peronosclerospora sorghi. The accumulation of MRNA in resistant lines was higher and lasted longer than that in susceptible lines. These results suggest that PAL and CHS may play a role in host as well as nonhost resistance to some fungi. No consistent differences were found between inoculated and control seedlings or among the treatments for CAD MRNA, suggesting that sorghum does not synthesize lignin as a strategy to prevent spread of these fungi.