Taxology And Relationships Analysis In Some Species Of Cycadceae By ISSR Markers

Cycadceae is a remote antique seed plant with highly value of viewing and science.At present,there is hot disputation about its taxonomic problem. In this paper,ISSR marker was used to study taxology and relationships of some Cycadceae species.Major results were as follows:1.The genomic DNA of Cycadceae with high quantity could be obtained with the improved method of CTAB which was good enough for ISSR amplification.2.A suitable ISSR-PCR reaction system was established effectively by orthogonal design and Single factor design methods.Namely 20μl reaction system contained:1×Buffer,75ng DNA,250μmol.L^-1dNTPs,1.0U Taq DNA polymerase,0.2μmol.L^-1Primer,2.5 mmol.L^-1Mg²⁺.Programmed for an initial step of 5 min at 94℃,followed by 40 cycles of lmin at 94℃,1min at 52℃and 2 min at 72℃,and a final 10 min extended at 72℃,saved in 4℃.3.11 ISSR primers which were chosen from 100 found to produce cleanly,repeatedly and stably polymorphic segments,and they produced 115 of 132 amplified fragments,the polymorphic ratio reach 87.1%,it explained the genetic distinction and relationships of 29 Cycadceae species effectively. The result of ISSR cluster showed C.longlinensis and C.segmentifida, C.longipetiolula and C.multifrondis,C.taiwaniana and C.hainanensis, C.revoluta and C.taidungensis,C.elonga and C.pectinata,the five groups had a near morphology and showed close genetic relationship. Zamiafurfuracea is one Zamiaceae species which showed having far genetic relationship with Cycadceae species;C.micholitzii had close genetic relationship with C.multipinnata but far genetic relationship with C.longipetiolula and C.multifrondis.It showed close distance having closer genetic relationship than far distance.4.The similarity coefficients of 29 species were ranged from 0.40-0.83 which indicated the material having obvious species difference.According to subspecies,varietas and forma that three ranks index coefficients,each cycad was established classification position:C.xilingensis and C.longiconifera was subspecies of C.segmentifida,C.segmentifida was original subspecies; C.longlinensis was varietas of C.segmentifida,C.segmentifida was original varietas;C.guangdongensis,C.taiwaniana and C.szechuanensis were independent species;C.hainanensis was varietas of C.taiwaniana and C.taiwaniana was original varietas;C.longipetiolula was independent species;C.multifrondis was forma of C.longipetiolula;C.longipetiolula was original forma;C.multipinnata was varietas of C.micholitzii,C.micholitzii was original varietas;the genetic difference of C.segmentifida and C.multipipinnata had reached variety level;C.brevipinnata and C.ferruginea were subspecies of C.miqueii,C.miqueii was original subspecies;C.parvula and C.balansae were independent species; C.shiwandashanica and.C.diannanensis were respectively variety and subspecies of C.balansae;cycadaceae species which were thought to have obvious difference in phenotypic character and have not any disputation in past time,but their similarity coefficients were high,reached the level of subspecies and varietas,there were no differences in some disputed cycadaceas species with those cycadaceas species which were thought to have clear classification position in genetic level.We had also given these species which had not any disputations in past some advice for their classification position,so that we could learn the relationship of every cycadaceae species well and draw some protective measure effectively.