ISSR Analysis Of Honeysuckle

honeysuckle is one of rare Chinese traditional medicine,and it has very important exploitation value.Unfortunately, there are many problems such as identification,classification,genetic diversities that affect the development of honeysuckle resources use. In this study,we inducted ISSR analyzing technique to analyse genetic diversities among 22 honeysuckle varieties come from HUNAN,SHANDONG,HENAN where is the mainly output area of honeysuckle.In order to generate useful information for identification,conservation and use of honeysuckle genetic resources.Major results were as follows:Effects on isolating honeysuckle genomic DNA by using SDS,CTAB, improved CTAB were analyzed and compared.One varietie L.macranthoides "golden-green" was used. The result showed that the three methods can obtain honeysuckle genomic DNA well.These three methods were ranked as:SDS,improved CTAB,CTAB according to the average yields of honeysuckle DNA in mass from high to low,and ranked as: CTAB,improved CTAB, SDS according to the honeysuckle DNA purities from high to low. These results suggested that honeysuckle DNA obtained by CTAB displayed superiority than others for molecular biological operations.By the usage of a single factor test, an optimized PCR reaction system for ISSR analsis was established:PCR was performed in a 20μL reaction mixture with 1×PCR buffer(Mg2+free),1.5UTaq DNA polymerase,0.15mmol/L dNTPs,0.4μmol/Lprimer,1.5 mmol/L MgCl2,60ng template DNA.The reaction process went as following:the temperature profile used for PCR was 94℃for 5 minutes,followed by 35 cycles of 94℃for 30 seconds,49.9℃for 30 seconds,72℃for 1.5 minutes,and was terminated with a 7-minute DNA extension step at 72℃.Through screening,obtained 10 primers from 100 ISSR primers.These 10 primers could amplify clear,stable,high genetic diversity bands.And 8 of them were two nucleotide acid repeats,account to 80%,especially (AC)n and (CA)n,account to 60%,and these two repeats amplified most genetic diversity bands.It showed that there were rich TG/GT repeats in honeysuckle genomic.In 22 experimental varieties,108 DNA bands were amplified by 10 ISSR primers,in which 96 bands were polymorphic,polymorphic ratio(PPB) was 88.9%.10.8 bands were amplified by each primer.The genetic similarity coefficients ranged from 0.41-1.00,and the average was 0.71. The dendrogram of UPGMA cluster revealed the relationships of individual about the 22 honeysuckle cultivars clearly,and The 3 dimensions plot of the principal coordiates analysis revealed the relationships of two populations about 22 honeysuckle cultivars.