Classification And Identification Of Citrus Maxima ’Tomentosa’ And Its Related Cultivars On Molecular Markers

Purpose:Hua Juhong,derived from cultivars of Citrus maxima’Tomentosa’or Citrus maxima（Burm.）Merr.,has the functions of curing diseases such as:cough,asthma,vomit,etc.It is a kind of sifted high quality medicine in China through the ages and one of the top ten southern herbs in Guangdong Province.Among them,Mao Juhong derived from cultivars of Citrus maxima’Tomentosa’is a traditional trueborn medicinal material,which has enjoyed a high reputation in Guangdong Province.There is a huge price margin between Mao Juhong and Guang juhong（derived from the other C.maxima）.However,it is difficult to identify the C.maxima‘Tomentosa’because of the complex of its original plant source.Although traditional identification methods on plants are often limited by many other factors of their growth year,organ and environment etc.,the method of molecular marker identification is based on the differences of the genetic material in plant with little influence by these factors,and its result is reliable,objective and accurate.Therefore,the aim of this study is to develop a rapid and effective molecular markers method to identify the C.maxima‘Tomentosa’and its related cultivars.We could discuss the genetic background within the different cultivars of C.maxima‘Tomentosa’.And we hope that the method can distinguish the Hua Juhong and Guang Juhong in seedling stage and facilitate future breeding programs,which could establish foundation to standardize the Hua Juhong marketMethod:In this study,we discussed the genetic polymorphism of C.maxima‘Tomentosa’and its related cultivars on 26 samples by using three barcode sequences:ITS2、psb A-trnH and matK,to find the barcode sequence suitable for C.maxima analysis.We used SCoT and SRAP makers on this study and combined two molecular makers to analysis these 26 samples’DNA.A rapid and intuitive SCAR marker was developed Based on the SCoT and SRAP markers for identification analysis to provide a rapid and efficient method for the identification of C.maxima‘Tomentosa’and its related cultivars.Result:1.Three international universal barcodes were used to study C.maxima‘Tomentosa’and its related cultivars.There was only one base mutation found in MatK sequence at the outer group samples,and we give it up on the following study.The ITS2 sequence showed higher mutation rate,and the mutation rate of psbA-trnH was lower than that of ITS2,but the clustering results shows the psbA-trnH is closer to morphology classification,and the clustering of psbA-trnH is better than the result of combined ITS2 and psbA-trnH.But all the 3 barcodes sequence is difficult to classify the plant within cultivars,especially in C.maxima analysis.2.We optimized SCoT-PCR reaction system by orthogonal design and single factor method.The optimization SCoT-PCR reaction system is:3 mmol/L Mg²⁺,0.3 mmol/L dNTPs,0.75 U Taq enzymes,0.6 umol/L primer and 40 ng DNA.6 primers were screened from 36 primers and annealing temperature optimization.94 bands were amplified from 26 samples,acquire average polymorphism rates of 73.4%.We construct the clustering tree by UPGMA to study the classification and identification of C.maxima‘Tomentosa’and its related cultivars.3.We optimized SRAP-PCR reaction system by orthogonal design and single-factor method.The optimal SRAP-PCR reaction system is:3 mmol/L Mg²⁺,0.25 mmol/L dNTPs,0.75UTaq enzyme,0.4μmol/L each primer and 40ng DNA.We acquired 7 primers through the screening of 110 pairs of primers.A total of 84 bands were amplified,with polymorphism rates of 70.24%.The clustering tree is similar to SCoT,but more accurate.The clustering tree which constructed based on the two markers was similar as the clustering tree constructed by SRAP,but more accurate.The result indicates that the SCoT and SRAP markers are suitable for C.maxima‘Tomentosa’and its related cultivars.Mantel test was compared the results of SCoT,SRAP markers and their combination,it showed that the similarity within the three markers at a high level,indicating that SCoT markers and SRAP markers could be effectively combined,and the results were reliable and accurate.4.Three specific bands were found in the study of SCoT markers and SRAP markers.We recovered these 3 bands and acquire its sequence by linked,transformed,cloned and sequenced.The specific primers were redesigned and tried to be transformed into SCAR markers.But we found a uniform band in all the samples at the verification of SCAR markers,indicating that the transformation of SCAR markers failed.We study on literature reports and experiments and summarized the several reasons that may lead to failure,hope our research could lays certain foundation for the later development of SCAR markers.Conclusion:Three DNA barcode sequences can’t completely identify C.maxima‘Tomentosa’and its related cultivars;only the psbA-trnH region was closer to morphology classification.The amplification efficiency and polymorphism of SCoT marker were higher than that of SRAP marker,but SRAP marker was closer to morphology classification in cluster analysis.Combined with the SCoT maker and SRAP maker,it can completely distinguish C.maxima‘Tomentosa’and its related cultivars.We developed the SCAR maker based on SCoT marker and SRAP marker.We obtained 3 specific sequences and designed specific primers for it.However,the polymorphism disappeared in the final verification and the transformation failed.Based on the experimental results and with reference to some related eassy,we summarizes some reasons that may lead to the failure of SCAR marker transformation,in order to provide some reference and lessons for the following research.