RAPD And ISSR Analysis Of Genetic Diversity Of Hovenia Acerba In Fujian

Hovenia acerba was a precious and economical specie, which could be timber, garden tree, or used by its fruits and in medical. Moreover, Hovenia acerba grew fast, had strong adaptability and high developing value. The research on genetic diversity was an indispensable part of resource development and utilization of Hovenia acerba.In this paper, the methods of RAPD and ISSR molecular markers were used to study genetic diversity and the genetic relationship of 35 Hovenia acerba germplasms from 12 provenances. The purpose was to provide a theoretical basis for the development and utilization of Hovenia acerba resources, selection of good germplasm and hybridization. Results were as follows:(1) The reaction system of RAPD and ISSR fitting for analyzing the genetic diversity of Hovenia acerba were established and optimized. The best RAPD reaction system was as follows: total reaction volume 25μl, 10×Ex Taq Buffer (Mg2 + plus) 2.5μl, dNTP Mixture200μmol/L, primer 0.5μmol/L, DNA template 25ng, Ex Taq enzyme 1.5U, ddH2O 17.95μl. Procedures: pre-denaturation 94℃5min, denaturation 94℃1min, annealing 36℃1min, extension 72℃2min, 45 cycles, final extension of 72℃10min. The best ISSR reaction system was as follows: total reaction volume 25μl, 10×Ex Taq Buffer (Mg2+ plus) 2.5μl, dNTP Mixture 375μmol/L, primer 0.4μmol/L, DNA template 75ng, Ex Taq enzyme 1U, ddH2O 14.55μl. Procedures: pre-denaturation 94℃5min, denaturation 94℃30s, annealing 50℃1min, extension 72℃90s, a total of 35 cycles, final extension of 72℃7min.(2) 10 RAPD primers and 10 ISSR primers were respectively selected, for they showed strong, reproducible amplification and distinct polymorphisms in 50 RAPD primers and 50 ISSR primers. 193 locus of genomic DNA in total were detected in 10 RAPD primers, of which there were 161 polymorphic locus, or, say 83.42%. The number of locus amplified by each primer was between 13 and 27, with an average of 19.3. The number of bands amplified by 10 primers in all materials was 3956. The number of amplified bands by each primer in each materials was between 3 and 19, with an average of 11.3. 175 locus of genomic DNA in total were detected in 10 ISSR primers, of which there were 155 polymorphic locus, or, say 88.57%, with 4 point more than that in RAPD. The number of locus amplified by each primer is between 15 and 23, with an average of 17.5. The number of bands amplified by 10 primers in all materials was 2910. The number of amplified bands by each primer in each materials was between 3 and 15, with an average of 8.3. Each amplified band was regarded as a character, and the binary data matrix was set out according to the existence of the bands. It's assumed that in the Hardy-Weinberg equilibrium, Na, Ne, I, H, Ht, Hs, Gst, Nm, Nei's(1972)genetic distance and genetic identity were calculated, and dendrograms were drew out based on Nei's (1972)genetic distance by the method of UPGMA.Analysis showed that, RAPD and ISSR molecular markers could reflect genetic differences and the genetic diversity among Hovenia acerba well. 35 measured materials from 12 provenances of Hovenia acerba showed abundant genetic diversity. The results would provide a theoretical basis in resources for development and utilization of Hovenia acerba in molecular level.