A New Method To Induce Chromosome Aberrations In A Triticale Cutivar Jinghui1 By Zebularine

Zebularine,(1-(β-D-ribofuranosyl)-1,2-dihydropyrimidine-2-one,is a cytosine analog with absence of synthetic 4-amino-pyrimidine ring.As a methyltransferase inhibitor,zebularine could specifically bind the methyltransferase and inactivate its function.Zebularine shows effects in reducing genome-wide DNA methylation level,reactivating silenced genes and chromosomal aberrations.In this study,a common wheat Huixianhong and Jingzhouheimai amphiploid Jinghui 1 developed by Nanjing Agricultural University was used to study the effect of zebularine on inducing chromosome aberrations in Triticeae crops,aimed at developing a new method to induce chromosome variations.The main results were as follows:By seed soaking with zebularine,the seed germination rate treated with 0,250,500 and 750 μM zebularine at 24 h and 24 ℃ was 45.0%,56.7%,50.0%and 46.7%,respectively.FISH analysis on mitotic chromosomes showed that cells with chromosome aberrations in four concentrations were 0%,36.8%,41.2%and 40.0%,respectively.The averaged chromosome aberrations in each cell at four treatments were 0,1.7,2.7 and 2.2,respectively.At 500 μM and 24℃,seeds treated for 12 h,24 h,36 h and 48 h showed the germination rate at 56.7%,58.3%,53.3%and 38.3%,respectively.Cells with chromosome aberrations were 36.6%,28.6%,23.5%and 54.0%correspondingly.The averaged chromosome aberrations per cell were 2.4,1.7,1.4 and 4.6 accordingly.In summary,seed soaking with 500μM zebularine for 24 h could effectively induce chromosome aberrations.From 98 M1 seeds,340(37.0%)cells with chromosome aberrations were observed,and 817 chromosome aberrations were obtained,which included six types with the frequency ordered as deletion and acentric fragments(49.0%)>large fragments translocation(28.9%)>small fragments translocation(17.1%)>intercalary translocations(2.6%)>dicentrics chromosome(1.9%)>ring chromosome(0.5%).Phenotypic analysis showed plant height,fertility and tillers showed obvious changes in M1 plants.After FISH analysis,45 M2 plants from M1 21 plants with phenotypic changes obtained in 2013 were identified and found four(8.9%)with chromosome aberrations,including one(2.2%)chromosome number variation and 3 plant(6.7%)chromosome structural variations.From 62 randomly selected M2 plant,20 with aberrations(32.3%)were identified,including 11 plant(15.9%)with chromosome number variations and 9 plant(14.5%)with chromosome structural aberrations.Analyzed with 14 rye chromosome specific molecular markers,from 58 randomly selected M2 plants,we found 22(37.9%)with absence of markers indicating chromosome aberrations occurred in these plants.The total absent markers were 36 with an average at 1.6,varied from 1-4.The study showed that zebularine could effectively induce chromosome aberrations and affect phenotypic traits.Combining analysis with molecular markers and FISH could increase the detection frequency of chromosome variations.The study also preliminarily studied the effect of tillering treatment using zebularine.12 chromosome aberrations from 72 M1 seeds were obtained,indicating tillering treatment might be another approach to induce stable aberrations using this chemical.