Development Of SCAR Marker For Tilletia Tritici

Common Bunt caused by wheat smut fungus is an infectious disease of wheats throughout the wheat-production regions of the world which causes yield loss and even no harvest.Besides,Tilletia spores contain trimethyla and other toxic substances that harm the human body.The fungus smut seedling wheat and the symptom are not obvious until the wheat matures.Because of that,it is very difficult to identify the early onset.Furthermore,it is hard to distinguish the three fungus(Tilletia tritici,Tilletia laevis,and Tilletia controversa)with the naked eye or microscope.Identifying make it crucial to develop molecular markers against different Tilletia spores.This study was carried out based on the former primers to Tilletia controversa and Tilletia laevis.We took Tilletia tritici as the research object and obtained the following results:1.Screend the specificity of ISSR primers to Tilletia triticiTilletia tritici,Tilletia laevis,Tilletia controversa were cultured,DNA-extracted and then PCR-amplified with ISSR markers.Then the bands were observed by gel electrophoresis to screen ISSR primers specific to Tilletia tritici.The results showed that the ISSR827 primer was the most specific to Tilletia tritici.2.Design SCAR marker primer Erc19 for the amplified band sequence of ISSR827 primerThrough preliminary experiments,the special bands of ISSR827 against Tilletia tritici.were cut and purified under UV lamp.The ligation vector was transferred into E.coli for sequencing and following primers designation.The results confirmed that the SCAR primer Erc19 designed according to the ISSR827 amplified sequence was specific to Tilletia tritici.3.Testing and optimize of SCAR marker primer Erc19In order to further Taq Mix,temperature,verify the specificity of SCAR marker primer Erc19.Test results for MIX applications showed that all three Taq MIX types produced specific amplification,with the best performance being the Acrid 2 X Pro Taq Master MIX(Dye Plus).The test results for temperature showed that the primer specificity was the best within the range of 58.5℃to 61.5℃,and the subsequent experiments showed that 61.5℃was the optimal temperature.we selected 11 common diseases on wheat,extracted their DNA and amplified with the primers.The results confirmed that among the 11 common diseases,the SCAR marker only produced specific amplification for Tilletia tritici.After that,we tested the sensitivity of SCAR-labeled primer Erc19.In the experiment,we diluted the DNA concentration of Tilletia tritici from100ng/μL to 1 pg/μL to amplify SCAR-labeled primer erc19.It was found that the marker at 1 ng/μL would still amplify bands for Tilletia tritici.4.Develop q PCR primers based on SCAR markers to improve the sensitivity of primersThe current development of q PCR technology has further requirements for the accuracy of identifying primers.Based on SCAR,we designed q PCR primers for the sequence and tested its sensitivity to draw amplification curve,dissolution curve,and standard curve to evaluate the label.Experimental results confirmed that the designed q PCR primers are specific to the plasmid connecting the target band at the lowest DNA concentration of 2.4fg/μLIn summary,these results indicate that the ISSR827 obtained by screening ISSR primers is specific to Tilletia tritici and it amplifies a specific band distinguished from Tilletia laevis and Tilletia controversa.The SCAR marker primer Erc19,which was further designed according to the sequence,has strong specificity to Tilletia tritici,and can also amplify bands when the DNA concentration is 1 ng/μL.In the next step,the q PCR primers designed based on the sequence are also specific to Tilletia tritici from the melting curve and amplification curve.From the standard curve,it maintains high sensitivity after dilution 10~5.This shows that it can still be detected when the primer plasmid concentration is diluted to 2.4 pg/μL.