| Hippophae rhamnoides subsp. sinensis, a dioecious and deciduous shrub species, occupies a wide range of habitats in the Wolong Nature Reserve, Southwest China. Our present study investigated the pattern of genetic variation and differentiation among five natural populations of H. rhamnoides subsp. sinensis, occurring along an altitudinal gradient that varied from 1,800 to 3,400 m above sea level in the Wolong Natural Reserve, by using ISSR and AFLP markers to guide its genetic improvement and germplasm conservation. And, comparative study of ISSR, AFLP and RAPD was performed to detect their capacity to estimating the level and pattern of genetic variation occurring among the five elevation populations of H. rhamnoides subsp. sinensis, and to discuss their application to the study on plant genetics. The results were list following:1. The ISSR and AFLP analysis conducted for the H. rhamnoides subsp. sinensis populations located in the Wolong Natural Reserve of China revealed the presence of high levels of genetic variation (h=0.249, H_T=0.305). Besides such features as relatively wide distribution, dominantly outcrossing mating system, and effective seed dispersal by small animals and birds, it is sometimes argued that hard climatic conditions and heterogeneous habitats may also contribute to high levels of diversity.2. Genetic diversity of H. rhamnoides subsp. sinensis populations was found to vary significantly with changing elevation, showing a trend that mid-elevation populations (2,200 m and 2,600 m) were genetically more diverse than both low-elevation (1,800 m) and high-elevation populations (3,000 m and 3,400 m). H. rhamnoides subsp. sinensis is thought to be stressed by drought and high temperature at low elevations, and by low temperature at high elevations. The high genetic variability present in the mid-elevation populations of H. rhamnoides subsp. sinensis is assumed to be related to a greater plant density in the middle altitudinal zone, where favorable ecological conditions permit its continuous distribution covering the zone from 2,200 m to 2,600 m above sea level.3. The genetic structure of H. rhamnoides subsp. sinensis revealed by ISSRs and AFLPs followed the general pattern detected in woody species with widespread distributions and outcrossing mating systems. Such plants possess more genetic diversity within populations and less variation among populations than species with other combinations of traits.4. In the present study, Mantel tests showed positive correlations between altitudinal distances and genetic distances among populations or subpopulations. The observed relationship between altitude and genetic distances, and the result of the cluster analysis including populations or male subpopulations and classifying the groups into three altitude clusters suggest that altitude is a major factor that restricts gene flow between populations and subpopulations.5. The analysis of molecular variance showed that only 3.8% of the variability resided between female and male subpopulations. Such a very restricted proportion of the total molecular variance between female and male subpopulations is due to common sexuality and mixing of genetic material between females and males. 6. The analysis based on ISSRs, AFLPs and RAPDs all revealed relatively high levels of genetic variation among different altitudinal populations of H. rhamnoides subsp. sinensis in Wolong Natural Reserve of China. Their estimates of mean Nei's gene diversity is equal to 0.249, 0.214 and 0.170 respectively, suggesting the higher capacity of detecting genetic variation of ISSR and AFLP than RAPD. It might be ascribed to their distinct sensitivity to different type of genetic variation.7. Based on the coparative study on ISSR, AFLP and RAPD, we drew a conclusion that they all successfully reveal some useful information concerning the level and pattern of genetic vatiation occurring among different elevation populations of H. rhamnoides subsp. sinensis. AFLP is a ideal tool to taxonomic study and individual identification for their highest efficiency index and marker index among the three marker systems.
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