| Cunninghamia lanceolata, a kind of arbor in Taxodiaceae, is an vital timber species for afforestation in southern China. The seed orchard is an important part of tree breeding system, of which genetic diversity determines the adaptability of seed and that affects the stability and multiple benefits of the forest. After Long-term natural and artificial selection, Cunninghamia lanceolata in different distributions produced larger genetic variation and formed different geographical groups under the influence of the climate and environment.The genetic diversity of the1.5and2generation clonal seed orchard and the phylogeography of Cunninghamia lanceolata of the whole distritution were studied, which revealed the population structure and the demographic history of Cunninghamia lanceolata, and provided information of molecular population genetics for the high generation genetic improvement.The main results are as follows:(1) The orthogonal design was used to optimize the amplification of the reaction system, and the suitable ISSR-PCR reaction system(20μl) was established as follows:1.5mMMgCl2,0.2mMdNTPs,0.7μMprimers,90ng DNA template,0.5U TaqDNA polymerase; the suitable PCR reactions were predenaturing at94℃for5min,40cycles of denaturation at94℃for30s, annealing at52℃-55℃for40s and extension at72℃for2min,with7min final extension at72℃and then saved at4℃.(2)10primers were screened to amplify108samples collected from1.5and2generation in clonal seed orchard of Cunninghamia lanceolata. A total of92polymorphic loci were amplified and117alleles were detected using ten primers. The average percentage of polymorphic loci was78.63%,the average Nei's genetic diversity of the two seed orchard was0.2314,the Shannon's information index was0.3575.Gst in the average was0.0830.Gene diversity within populations account for91.7%and between populations8.3%.Most variation in Cunninghamia lanceolata was found within rather than among populations. The percentage of polymorphic of the1.5and2generation in clonal seed orchard were67.52%and60.68%. the effective number of allele per locus were1.3507and1.3627, the Shannon's index were0.3252and0.3172,Nei's index were0.2126and0.2118. The results implied a quite wide genetic basis and rich genetic diversity in the seed orchard. The genetic diversity of the2nd generation seed orchard was slightly lower than the15,but still maintain a wide genetic basis.(3) Three cpDNA fragment(petD,trnV-tmM and psbA-trnrm) were sequenced for297trees of16populations. A total of nine cpDNA haplotypes were identified, one ancestral haplotype(HapA) was widely found in each population, six haplotypes(HapD,HapE,HapF,HapG,HapH,Hapl) occurred only in one population as population-specific.The phylogenetic tree of chloroplast haplotyes was contructed by maximum parisomy. Nine haplotypes were divided into only two:HapA as an independent one,the last several merged into one.Genetic differentiation among populations(Gst)was0.050,Nst was0.046,the average gene flow(Nm) was0.418.The analysis of molecular variation(AMOVA) indicated that91.86%of total variation was partitioned between the populations, whereas8.14%among population, the fixation index was0.0814.The result indicated the genetic variation mainly occurred within populations and that the genetic structure of cpDNA variations was not obvious.(4) With the central network, we preliminary think, the haplotype frequency in Anhui and Fujian province is low,while in Zhejiang,Hunan,Guangdong and Jiangxi province is much higher. The result indicated that Cunninghamia lanceolata migrated to lower altitude where had a smaller impact of the glacial times and that during interglacial period, there may be bottleneck effect in the demographic history of Cunninghamia/anceo/ata. Combined with the published fossil research literature, the molecular phylogeographic study result of Cunninghamia lanceolata showed that the distribution of Chinese fir may not be its origin,but the differentiation zone or relict central area. |
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