Barley TILLING Population And Screening Of Mutants In JA Signaling Pathway

TILLING (Targeting Induced Local Lesions In Genomes) is a powerful reverse genetictool widely used in many important crops. This study constructed an EMS-based mutantpopulation on the barley genotype Tamalpais and developed the CEL I-based TILLINGscreening system. The system was used to identify the mutant alleles of AOC, AOS and COI1genes that are potentially involved in the jasmonate acid (JA) signaling pathway. The barleyTILLING population will be used as a powerful tool to study the functional genomics inTriticeae crops. In detail, the study includes three sections.1. The development of barley TILLING population. We used29mM EMS solution totreat mature kernels. The whole population including22,567individuals was generated inthree phases. The phase one subpopulation contained2,154M2lines. The phase twosubpopulation had10,389M2individuals;6.21%of the tested seedlings displayed variationsin leaf chlorophylls in the greenhouse. In field, the phase two subpopulation showed abundantphenotypic variations, such as those in creeping growth habit, number of tillers, plant height,flowering time, leaf color, leaf shape, leaf stripe, leaf spot, spike characteristics, and sexualreproduction. In particular, mutation rates for creeping growth habit, number of tillers, plantheight, leaf color, leaf stripe, and leaf spot were0.11%,6.03%,0.13%,2.5%,0.18%, and0.17%, respectively. Sampling survey revealed that the population was low on embryolethality. About9%of surveyed M2individuals had rates of embryo lethality over50%. Thephase three subpopulation contains10,024M2individuals, and the subpopulation displayedhigh levels of phenotipical variation. The Tamalpais population will play roles for functionalstudies in Triticeae crops. 2. The optimization of CEL I digestion. The CEL1enzyme that nonspecifically cleavesthe mismatch in DNA double strands is an important component of the TILLING screeningsystem. In this study, active CEL1enzyme was successfully purified from the local growingcelery in Tai’an, China. Plasmids associating with single mismatch was used as PCRtemplates. The CEL1-based cleavage and the PAGE-based detection were employed todevelop the TILLING system. Factors of CEL1amount, digestion time and tempature, and thecleavage buffer were considered during the optimization process. The CEL1enzymeeffectively cut the mismatch DNA after30-min incubation in a0.1×cleavage buffer (20mmolL-1HEPES pH7.5,10mmol L-1KCl and3mmol L-1MgCl2) under45℃.3. Mutant screening in the JA signaling pathway. Orthologs of barley AOC, AOS andCOI1were identified. Corresponding mutants were obtained in the current TILLINGpopulation. Twenty-two mutations were discovered, and the mutation frequency is about1point mutation in every303Kb region.This study developed a large Tamalpais TILLING population, and mutations of the JApathways genes such as AOC, AOS and COI1were obtained. This study sets solid foundationfor developing reverse genetics tool in Triticeae Crops.