Sun, Qizhong

Yang, Xiufang

English

University of Kentucky

University of Kentucky UKnowledge International Grassland Congress Proceedings XXI International Grassland Congress / VIII International Rangeland Congress Application of ISSR‐PCR to Identification Two Different Ecotypes of Lespedeza hedysaroides Xiufang Yang Gansu Agriculture University, China Qizhong Sun Grassland Research Institute, China Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Plant Sciences Commons, and the Soil Science Commons This document is available at https://uknowledge.uky.edu/igc/21/12-2/26 The XXI International Grassland Congress / VIII International Rangeland Congress took place in Hohhot, China from June 29 through July 5, 2008. Proceedings edited by Organizing Committee of 2008 IGC/IRC Conference Published by Guangdong People's Publishing House This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in International Grassland Congress Proceedings by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. 　 Multifunctional Grasslands in a Changing World 　 Volume Ⅱ 　 瞯 ]407　 瞯Grasslands/Rangelands Production Systems——— Developing Improved PlantsApplication of ISSR‐PCR to identification two different ecotypes of Lespedeza hedysaroidesY ang X iu‐f ang1 , Sun Qi‐z hong21 College o f G rassland Science ,Gansu A griculture University ,L anz hou ,Gansu Prov ince 730070 ,China , yy rrmm123＠ 126 .com ; 2 G rassland Research Institute , Inner Mongolia ,Hohhot 010010 , China , sunqz＠ 126 .comKey words :Lespedez a hedysaroides ,ISSR‐PCR ,molecular marker ,identificationIntroduction Inter‐simple sequence repeats‐polymerase chain reaction ( ISSR‐PCR) is a new type of molecular marker technologyin recent years ( Zietkiewiez , １９９４) . Universal primers were designed according to the genome of microsatellite sequences forthe genomic DNA PCR amplification . ISSR‐PCR has been widely applied variety identification , fingerprint mapping and geneticdiversity analysis . The objective of this research was to identify two different ecotypes of L . hedysaroides ( the light green leafand the dark green leaf ) by Zhao Yang etc . ( Zhao Yang , ２００６) Lespedez a spp . ISSR‐PCR reaction system optimized .Materials and methods Materials came form the test area in Linxi county of Inner Mongolia . Collected barley leaves stored inliquid nitrogen , then stored at ‐２０ ℃ . The genomic DNA extracted by the extraction kit . In ２０μL reaction mixture are asfollows : ４０ ng genomic DNA , ０ .２μmol L‐１ ISSR primer , １０μL ２ × Taq PCR MasterMix ( ０ .１UμL‐１ Taq Polymerase , ５００μMdNTP each , ２０mM Tris‐HCL( PH８ .３ ) , １００ mM KCL , ３ mM MgC１２ ) . The PCR procedure is one preliminary denaturation at９４ ℃ for ５ min ; ３５ cycles each involved denaturation at ９４ ℃ for ３０ S ,anneal at ５０ ～ ５６ ℃ ( decided by primer ) for ４５ S ,ex tended at ７２ ℃ for １ min ３０ S ;and a final extension at ７２ ℃ for ７ min , then keep the temperature at ４ ℃ . The PCR productswere examined by electrophoresis with ２ .０％ agarose gel .Results Among the eight primers ( Table １) , the primer UBC‐８８０ was able to distinguish the examined two different ecotypes ofL . hedysaroides ( Figure １) . There is a clear and obvious band in ５００bp .　 Figure 1 ISSR‐PCR p ro f iles o f two di f f erentecoty pes o f L . hedysaroides M indicates DNAstandard molecular weight . 1‐the dark green lea f ; 2‐the light green lea f .Table 1 Used ISSR p rimer sequences .primer sequenceUBC‐８１２ 湝(GA) ８ AUBC‐８０９ 湝(AG) ８GUBC‐８１１ 湝(GA) ８CUBC‐８２５ 湝(AC) ８ TUBC‐８２７ 湝(AC) ８GUBC‐８８０ 湝(GGAGA) ３ 骀UBC‐８４２ 湝(GA) ８YGUBC‐８２４ 湝( TC) ８GConclusion ISSR‐PCR provides a quick , reliable molecular marker technique for identification of two different ecotypes of L .hedysaroides . This method can be used in intraspecific identification of forage .ReferencesZietkiewiez E ,Rafalski A and Labuda D ( １９９４ ) Genome fingerprinting by simple sequeneerepeat ( SSR) .anchored polymerasechain reaction amplification .Genomies ,２０ :１７６‐１８３ .Zhao yang ,Chen Xiao‐yang &Li Tong‐sen etc .２００６ .Establisment and Optimization of ISSR Reaction System for Lespedez aspp . ,Journal o f Southwest Forestry College ,２６(２) ,６‐９ .

Application of ISSR‐PCR to Identification Two Different Ecotypes of \u3ci\u3eLespedeza hedysaroides\u3c/i\u3e

https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=3331&amp;context=igc

Application of ISSR‐PCR to Identification Two Different Ecotypes of \u3ci\u3eLespedeza hedysaroides\u3c/i\u3e

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