Generation Of A Barley TILLING-population And Its Use To Study The Diease Resistance Genes

TILLING (Targeting Induced Local Lesions In Genomes) is a powerful reverse genetic tool widely used in many important crops. This study developed an EMS-based TILLING population on the barley genotype'Tamalpais'and a CEL I - based screening system. The system was used to identify mutant alleles of the EDR1 and NPR1 genes that are potentially involved in the salicylic acid (SA) signaling pathway. The barley TILLING population will be used as a powerful tool to study the functional genomics in Triticeae crops.In detail, the study includes four sections.1. The development of the barley TILLING population. Pilot experiments were carried out to optimize the EMS treatment according to the germination, survival and albino rates of the M1 seeds or seedlings. The ideal protocol was to shake barley seeds in 3% (v/v) EMS solution for 10 hours under 25℃. The protocol was used to develop 2154 M₂ lines on the barley genotype'Tamalpais'. DNAs of every four M₂ lines were pooled together, and 534 DNA pools were created.2. The purification of the CEL I restriction enzyme. The CEL I enzyme that nonspecifically cleaves the mismatch in DNA double strands is an important component of the TILLING screening system. Since the commercial CEL I is very expensive, many labs prefer to purify CEL I from Celery. In this study, active CEL I enzyme was successfully purified from the local growing celery in Tai'an, China.3. The optimization of the TILLING screening system. Two DNA plasmids that contain only one single nucleotide polymorephism were used as the PCR template. The CEL I - based cleavage and the PAGE-based detection of the DNA mismatch were employed to develop the TILLING system. Factors of CEL I amount, digestion time and tempature, and the cleavage buffer were considered during the optimization process. The CEL I enzyme effectively cut the mismatch DNA after 30-min incubation in a 0.1×cleavage buffer (20 mM HEPES pH 7.5, 10mM KCl and 3mM MgCl2) under 45℃.4. The identification of gene mutantions related to the SA pathway. EDR1 (GenBank: AF305913) and NPR1 (ATU76707) are two crucial genes related to the SA pathway in Arabidopsis. Orthologs of barley EDR1 (AF305912) and NPR1 (AM050559) were identified. Corresponding mutants were obtained in the current TILLING population. Five mutations were discovered, three for EDR1 and two of NPR1. Two mutations occurred in the intron region; the rest three mutations included one silent mutation of the NPR1 gene and two missense mutations (His351Tyr and Pro556Ser) in the EDR1 gene.