Establishment And Analysis Of EMS Mutant Library In Oat(Avena Nuda L.)

Oat is an important crop for grain and forage. It is importance of building oat EMS mutant library for oat functional genomics studies and genetic improvement. So, in this study, chemical mutagen ethyl methane sulfonate was used to treat oat variety Huazao 2.We investigated the M2 plants during growth period, and detect mutant strains. At the same time, we also identified the genetic differentiation of mutants with molecular tools. The main results are as follows:(1) Seeds of Huazao 2 were treated for 15 h with 0.7% solution of chemical mutagen Ethyl methane sulfonate(EMS). A total of 4083 M1 plants were obtained. Then 2000 individual plants were gown in M2 generation. The morphological variation of plants was investigated in the growing season. Again some yellow mutants were planted in M3 generation for observing the genetic stability of the trait. Results showed that phenotypic variation is abundance after EMS treatment. 196 plants with different types of variation were found in the M2 generation. The mutation rate reached about 9.8%. The types of variation were very ample, including seedling habit, leaf color and shape, tiller number, plant height, spike morphology and maturity. Among these mutants, yellow leaf mutations properties can be stably inherited in M3 plants.(2)TILLING detect of oats mutant library. Crude of CEL I endonuclease was extracted from celery. Templates containing the different single fragments were used to detect the activity of extracted CEL I, it illustrated that it has higher activity. We explored cleavage reaction conditions, such as temperature, digestion time, the amount of the extract and others, and construction of the detection system. Using the system, two loci related to yellow leaf seedling mutants were identified.(3) Molecular characterization of mutant library. 10 pairs of SSR primers were used to detect 288 pools, and 72 loci were identified. The frequency of mutation is about 1/20 kb.98 pairs of specific primers. Base on the gene sequence of etiolation and stem height of rice, six pairs of primers were designed to amplify partial mutants identified in M2 generation. Detection of amplification products was taked with HRM and 25 loci were identified from mutants. These mutant samples were cloned and sequenced. Results showed that DNA sequences of loci in 19 individual samples were varied. Compared with BLAST of sequencing fragments, it is found that 4 samples had chl G gene, which is synthase for chlorophyll in oat, and only differented in single-base from wild type. The yellow leaf mutants M0368、M0782、M1888 and M1910 were planted for M3 generation, which approved that this property can be inherited stably.This study had established the EMS mutagenesis population and identified various mutant types in oat varity Huazao 2. The property of yellow leaf can be inherited stably, which will provide a basic material for further study of functional genomics and genetic improvement of oat.