Development Of A Jing411Wheat Mutant Library For TILLING Analysis

TILLING is a high-throughput screening method for fast and efficient identification of multiple genesand multiple traits in large populations to improve the efficiency of mutation screening. We built amutant population of wheat variety Jing411. Point mutations in quality-related genes of starch synthesispathway, glutenin subunits, grain hardness, etc. were tilled and some mutant phenotypes were alsoidentified. The main results were as following:1. Single-seed descent population with1245M2plants was established by using0.5%、1.0%and1.5%EMS for4h and bulk seed population with1040M2plants by using0.9%and1.5%EMS for4and8h. Field phenotyping were carried out during whole growth period. In the two populations36mutation types were found and the total mutation rate was13.44%. Mutation rate in Single-seeddescent population and bulk seed population was12.53%and14.52%respectively.2. We have identified193point mutations which include114alleles in9target genes in9DNA poolswhich were from the M2population. The average mutation density was1/122.75kb. Starch synthesisgenes include SSII-A、SSII-B、Agp2、Waxy and Sbe1have been detected by TILLING,93pointmutations with75alleles were found and the average mutation density was1/137.39kb.Five plantswere obtained which starch contents were significantly（α=0.01） lower than the control plant becauseof the point mutations in SSII-A gene.Four plants were obtained which were significantly different withthe control plant（α=0.01）in Agp2gene.3.37point mutations with33alleles were screened in Pina and Pinb genes. The average mutationdensity was1/164.43kb. The point mutation which changed the amino acids induced the increase ofseed hardness.22and40mutations were also found in GluD3-23and TEF1genes. The mutationdensity was1/52.07kb and1/31.84kb respectively. Two plants were found in GluD3-23and TEF1genewith lower grain protein content and starch content respectively（α=0.01）.4. Classify all point mutations according to the base changes. Transition included G-A and C-Tmutations was the most with the percentage49.74%. The percentage of transversion, heterozygous andinsertion was21.76%,15.03%and10.36%, respectively. Deletion was the least whose percentage was3.11%. Classify all point mutations according to the impact of amino acids. Intron mutations were themost with the percentage38.86%. The percentage of mis-sense mutation is37.31%. Synonymousmutation was less and the percentage was23.83%.Phenotypic test on some of the tilled point mutations showed thatcorresponding phenotype could bechanged significantly in plants because of the missense and nonsense mutations in target genes.It wasconcluded that the EMS induced population of wheat variety Jing411had diverse phenotypic mutationsand higher mutation frequency and could be the TILLING population of target genes.