Karyotype And ISSR Analysis Of Some Wild Lilium

This paper has adopted two methods to analysis the genetic relationship of the collected wild lily species. In the first part, karyotypes of the collected 14 species of wild lily were analysised, and then used clustering to analysis their genetic relationship. In part two, added another six unidentified wild species to earlier collected 14 lily species, and used ISSR maker to analysis the genetic relationship of the 20 lily species. The main conclusions were as follows:1. The nuclear-type in commonThe lily species has large chromosomes, basic number x= 12, most of them are diploid (2n=2x=24), only L.lacifolium Thunb was triploid (2n=3x=36). The Karyotypes of L.lacifolium Thunb, L.pumilum DC., L.davidii Duchart re were 3A that of others were 3B. The AK% of These three lilies were higher, which indicating that the Karyotypes of these lilies were more evolved, in the plant evolving system they may be in a derivative or more evolutionary status; Mostly, chromosomes consisted of two pairs of middle or near the middle long metacentric chromosomes and 10 pairs of near or telocentric chromosome, and in the first pairs of chromosomes often can saw a pair of intermediary satellite chromosomes.2. karyotype diversityThe karyotypes of 14 studied lily species had a rich diversity. Mainly manifested in differences in the composition of chromosomes, the number of satellite chromosomes and their distribution, and the existence of the B chromosome and their types.According to the differences of the chromosome composition, the 14 lilies were divided into 3 categories, namely:(1)lilies with m, sm, st, t chromosomes, including Llacifolium Thunb, L.rosthornii Diels, L.brownii F.E.Brown ex Miellez, L.leucanthum (Baker) Baker, L.pumilum, L.regale Wilson, L.longiflorum, L.davidii Duchartre.var.unicolor (Hoog.)Co Hon, L.taliense Franch, L.henryi Baker.(2)lily with m, sm, t chromosomes:L.tsingtauense Gilg.(3)lilies with m, st, t chromosomes, such as L.sargentiae Wilson, L.davidii Duchart re, Lsulphureum Baker.All the studied wild lilies had a pair of intermediary satellite chromosomes on the first pair of chromosomes except L.sargentiae Wilson, L.tsingtauense Gilg, L.longiflor-um; several lilies had satellite chromosomes on the long arm of the 11th pair of chromosomes; they were L.leucanthum (Baker) Baker, Ltsingtauense Gilg, L.davidii Duchartre.var.unicolor (Hoog.) Co Hon; L.pumilum had intermediary satellite chromos-omes on the 1st,6th,8th pair of chromosomes; L.tsingtauense Gilg had 2 pairs of satellite chromosomes which located on the long arms.In the studied 14 wild lilies, L.henryi Baker had three kind of number of B telocentric chromosomes, which were 1,2, and 6. L.leucanthum (Baker) Baker had a near metacentric B chromosome.3. karyotype evolutionAccording to Levitzky-Stebbins on karyotype evolution of flowering plants, the symmetry-the original, asymmetric-evolutionary point of view, the 14 lilies by karyotype asymmetry coefficient from high to low as fallow:L.sulphureum Baker＞L.laci-folium Thumb＞L.longiflorum＞L.sargentiae Wilson＞L.taliense-Franch.＞L.regale Wilson＞L.brownii F.E.Brown ex Miellez＞L.rosthornii Diels＞L.henryi Baker＞L.davidii Duchart re＞L.pumilum＞L.leucanthum (Baker) Baker＞L.tsingtauense Gilg＞L.davidii Duchartre.v-ar. unicolor(Hoog.)Co Hon.and this also indicated their evolution status.4. Cluster analysis and genetic relationshipAccording to the clustering results, the14 lilies were divided into four categories: first class:L.lacifolium Thunb; the second category:L.henryi Baker; the third category: Ltsingtauense Gilg, Lpumilum; the fourth category:L.davidii Duchartre.var.unicolor-(Hoog.)Co Hon, L.regale Wilson, L.taliense Franch, Lsulphureum Baker, L.longifloru-m, L.brownii F.E.Brown ex Miellez, L.sargentiae Wilson, L.leucanthum (Baker) Baker, L.davidii Duchart re, L.rosthornii Diels.5. In this study founded that a modified CTAB method which only 0.2g of fresh leaves was needed and added 2% PVP and 2% B-mercaptoethanol in the CTAB can get a higher concentrations, higher purity DNA, which was suitable for ISSR analysis of lily.6. Based on an orthogonal test with 3 levels of 4 factors (DNA, primers, Mg²⁺, Taq enzyme), picked up the combination which produced the clearest and most bands. And then on the basis of it, adjusted the level of factors, then came to the conclusion of the most excellent PCR reaction system:10×PCR buffer buffer 2.5ul,2.5mm/ldNTP 2.0ul,25mM/L of magnesiumions 1.5ul,5um/L primers 1.0ul, template DNA 10-20ng (0.3ul) taq enzyme 0.3ul(1.5U),17.4ul double-distilled water.Through trial and error, I got the optimized procedure:94℃5min,94℃45s, annealing temperature suitable for 45s,72℃75s,40 cycles,72℃8min,25℃, end.7. the six selected primers amplified 180 polymorphic bands, cluster analysis found that the analysis of 20 lily samples can be divided into five broad categories: first category:L.sargentiae Wilson, unidentified Lily, L.leucanthum(Baker)Baker, unidentified 4, L.davidii Duchart re, L.regale Wilson, L.davidii Duchartre.var.unicolor-(Hoog.)Co Hon.; second category:L.lacifolium Thunb, unidentified 3; the third category:L.tsingtauense Gilg, L.sulphureum Baker, L. longiflorum, L.brownii F.E.Bro-Own ex Miellez, L.taliense Franch, unidentified 2, L.henryi Baker; fourth category: L.pumilum; fifth categories:L.dauricum Ker.gewl., unidentified 1.8. The nuclear-cluster clustered the 14 lilies into four categories; ISSR cluster clustered the 20 lilies into five categories. The results of these two methods were very similar, most of the lilies in the group of sect.lilium in morphological classification were clustered into categories, but the lilies in the group of sect.sinomartagon were relatively decentralized. Nuclear clustering had no the lilies of the sect.lophophorum, but in the ISSR clustering, L.dauricum Ker.gewl which is a member of the sect.lophophorum was clustered into a separate category. In nuclear cluster L.tsingtauense Gilg in the group of sect.sinomartagon was categorized earlier, but in ISSR clustering, it was clustered into the category of sect.lilium. The differences between the results of two clustering may be due to the possibility that the mutation of the chromosomes morphology occurred, but did not in the gene level, or the gene level mutated, but chromosome morphology not. In real category, you can take the morphological classification, karyotype analysis and ISSR into account.