| To getting the number and morphology of chromosomes of China’s major Hemerocallis 5pp.germplasm resources,and carry out the research on Hemerocallis’ cross breeding,this reaserch used some methods such as technique optimization system、karyotype analysis and flow cytometry(FCM)and so on for the collection of Hemerocallis spp.germplasm resources to study the ploidy identification n chromosome structure and genetic diversity.Basis on these results that will be conducive to China’s Hemerocallis germplasm resources genetic research on cytology.(1)This reaserch used H0003 root material as material and analyzed the influence of 0.002mol/L 8-hydroxyquinoline solution pretreatment temperature and time,dissociation time,the soaking time in distilled water after dissociated and other factors.The results showed that the optimum pretreatment temperature and time of the 0.002molL-1 8-hydroxyquinoline solution were 4 ℃ and 4～6 h;the optimum dissociation time of 1 molL-1 HCI was 7～8 min,the optimum soaking time in distilled water after dissociated was 10 min,and the Carbol fuchsin stained 15 min.(2)Based on the above system,we identified 82 materials,and optimized the root tip chromosome preparation technology for 66 materials.The result showed that 53 materials were diploid(2n=2x=22),and 10 materials were triploid(2n=3x=33),and 3 materials were tetraploid(2n=4x=44).In the meanwhile,flow cytometry(FCM)was untilized to identified 26 materials with H0006(2n=2x=22)as the control material,the results showed that 14 materials were diploid(2n=2x=22),4 materials were triploid(2n=3x=33),8 materials were tetraploid(2n=4x=44).Above those materials,H0006、H0020、H0024、H0060、H0076、H0089、H0091、H0093 and H0144 these 10 materials were used two kinds of methods,and the result consistent.(3)34 different Hemerocallis spp.materials were analyzed by karyotype analysis,the results showed that 2A、IB、2B、3B four types of karyotype in 35 materials.Among them,H0025 and other 5 materials were 2A、H0051 and HO 130 were IB、H0001 and other 22 materials were 2B,H0006 and other 22 materials were 3B.The karyotype analysis showed that the chromosomes of 34 materials to median region(m)and submedian region(sm),and asymmetrical karyotype coefficient between 57.44%-68.40%.Among them,H0002,H0085 and H0118 were the same karyotype formula、H0003 and H0058 have the same karyotype、the karyotype formula of H0005,H0099 as same as H0121、H0006,H0018,H0088 and H0111 were the same karyotype formula、H0017 and H0065 have the same karyotype,H0027 as same asH0042、H0039,H0062 and H0097 were the same karyotype formula、the karyotype formula of H0069,H0106 as same asH0130(4)To Self cross,selfing and backcross between H0006(2n=2x=22)、H0026(2n=3x=33)and H0056(2n=4x=44).H0006(♂)XH0006(♀)and H0056(♂)×XH0026(♂)two combinations were 50 flowers pollination leave each otner,the result showed that ovary swelling rate were 48%and 26%,and the fruit set both was 4%.H0006(♂)× H0026(♀)was 100 flowers pollination,and the ovary swelling rate was 57%,and the fruit set was 46%.Above them,only the H0006(♂)× H0026(♀)combination seedling establishment,and the seedling rate was 71.61%(5)Under preliminary true and false identification of the H0006(♂)× H0026(♂)combination hybrid hybrids.Through 19 pairs of EST-SSR primers which showed polymorphism in their parents,were detected by polyacrylamide gel electrophoresis.Above them,3 pairs of primers showed paternal specific bands in the H0006(♂)bands,which could be used to identify the hybrids.The results showed that 10 hybrid progenies were preliminarily identified as true hybrids,with a proportion of 21.74%. |
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