ISSR Reaction Optimization And Genetic Diversity Analysis Of Eleusine Indica (L.) Gaerth In Part Of China

Goosegrass （Eleusine indica （L.） Gaerth） is an annual grass weed, with a strong vitalityand fecundity. It is regarded as one of the worst weeds. In this study, inter-simple sequencerepeat（ISSR）method was applied to detect genetic variation of Eleusine indica from6provinces, and genetic structure between and within the eighteen populations, to study theeighteen populations to reveal genetic diversity of Eleusine indica. The ISSR molecularmarker results were as follows:1. The orthogonal and one-factor experimental design design were applied to optimizeISSR amplification system at four levels of five factors(Mg²⁺, Taq polymerase, dNTP, primerand DNA template) respectively. A optimal reaction system was established,25μL reactionsystem contained DNA80ng、Mg²⁺1.75mmol/L、dNTP0.4mmol/L、primer0.3μmol/L、Taqpolymerase1U and10×PCR Buffer2.5μL. Amplification program included preodenaturationat94℃for5min, denaturation at94℃for30s, annealing temperature for1min, after35cycles, the product was extended at72℃for10min and kept at4℃.2.14polymorphic primers screened from30ISSR primers were applied to study thegenetic diversity of18Eleusine indica populations. Results showed that there were158fragments obtained by amplification of ISSR with14polymorphic primers and each primersgenerated10DNA fragments which molecular weight was between150²000bp. Amongthem,146fragments were polymorphic and the polymorphic rate reached at92.41%.3. The Jaceard coeffieient was calculated using NTSYS-PC2.10software. The resultshowed that similarity coefficient of the18populations varied from0.3125⁰.8750. l Thehighest genetic similarity co-efficient （0.8125） was observed between HS and ST, whichillustrated genetic exchange frequently and a closer relationship among above.4. The fingerpring data were further analyzed using UPMGA Cluster methods, whichclustered the18populations into3main distinct groups, the first cluster comprising all theaccessions of G, ZJ and ZY while the second cluster included five samples viz, BH, BF, HA,SW and SJ. The third cluster included another four samples viz, HC, HK, HS, ST, HX, HZ,AF, AB, SY and SY-2J. which indicated that there were some relations of genetic lineages ofEleusine indica to its geographical origin, despite the continued generation of the herbicidechoice, still maintained a high genetic diversity.