卧龙自然保护区不同海拔的中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传多样性分析

中国沙棘是一种雌雄异株、风媒传粉的灌木或乔木，在中国西南的卧龙自然保护区有广泛的分布。本研究以采集于四川卧龙自然保护区5 个海拔(1800 m、2200 m、2600 m、3000 m、3400 m)梯度的中国沙棘天然群体为材料，以ISSR 和AFLP 标记技术研究其遗传多样性水平及其遗传结构，旨在了解卧龙地区中国沙棘天然群体的遗传多样性水平以及遗传多样性在群体间、群体内以及雌雄亚群体间的分布和特征，为中国沙棘树种的遗传改良及种质资源保存提供遗传研究背景与实验依据。同时探讨ISSR、AFLP 和RAPD三种标记对中国沙棘天然群体的遗传变异水平和群体间遗传结构的评估能力和各自的优缺点。研究得出以下主要结论： 1. ISSR和AFLP分析都表明卧龙自然保护区的中国沙棘群体拥有较高的遗传变异水平(h = 0.249，HT = 0.305)。出现这种结果的主要原因可能与卧龙自然保护区多变的气候条件和生境的异质度大有关。 2. ISSR 和AFLP 都揭示出卧龙自然保护区中国沙棘群体的遗传多样性随着海拔的增加发生显著的变化，表现为中海拔群体(2200 m 和2600 m)比高海拔群体(3000 m 和3400 m)和低海拔群体(1800 m)有更高的遗传多样性的趋势。出现这种趋势的可能解释是低海拔群体处在相对高温和相对干旱的环境，高海拔群体受到低温和紫外线胁迫，而中海拔群体存在中国沙棘生长的适宜环境。 3. ISSR 和AFLP 分析都表明：卧龙自然保护区中国沙棘的遗传结构遵循分布范围广、交配系统以异交为主的木本植物的通常模式，即大多数的遗传变异存在于群体内，只有少部分的遗传变异存在于群体间。 4. 经Mantel 检测表明，卧龙自然保护区中国沙棘群体间的海拔距离和对应遗传距离之间存在显著的正相关关系，即随着垂直海拔距离的增加，群体间的遗传距离也随之增加。Mantel 检测结果以及聚类分析将卧龙自然保护区5 个不同海拔的中国沙棘群体分为低、中、高海拔群体三组的研究结果都表明，海拔很可能是限制群体间基因交流的主要因素。 5. ISSR 分析发现同一海拔的雌雄亚群体首先聚类的研究结果表明，同一海拔的雌雄亚群体在遗传上最相似。方差分析结果表明只有3.8%的总遗传变异存在于雌雄亚群体间，这可能与雌雄植株间的交配和遗传物质的混合有关。 6. ISSR、AFLP 和RAPD 分析都表明卧龙自然保护区不同海拔的中国沙棘天然群体的遗传多样性水平较高。它们的分析结果估算得到的Nei's 平均基因多样度(h)分别为0.249、0.214 和0.170。从该结果可以看出ISSR 和AFLP 比RAPD 检测到更多的遗传多态性,这很可能是不同标记检测的基因组的位点不同所致。 7. 依据对不同标记系统的比较分析，认为ISSR、AFLP 和RAPD 三种分子标记系统都能成功地用于调查卧龙自然保护区不同海拔的中国沙棘群体的遗传变异水平及遗传变异结构，提供关于中国沙棘天然群体多态性水平和遗传变异分布的有用信息。在三者中，AFLP 具有最高效能指数和标记指数，在确定种间分类关系或鉴别个体方面是一种比较理想的标记。 Hippophae rhamnoides subsp. sinensis, a dioecious and deciduous shrub species,occupies a wide range of habitats in the Wolong Nature Reserve, Southwest China. Ourpresent study investigated the pattern of genetic variation and differentiation among fivenatural populations of H. rhamnoides subsp. sinensis, occurring along an altitudinal gradientthat varied from 1,800 to 3,400 m above sea level in the Wolong Natural Reserve, by usingISSR 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 toestimating the level and pattern of genetic variation occurring among the five elevationpopulations of H. rhamnoides subsp. sinensis, and to discuss their application to the study onplant genetics. The results were list following: 1. The ISSR and AFLP analysis conducted for the H. rhamnoides subsp. sinensispopulations located in the Wolong Natural Reserve of China revealed the presence of highlevels of genetic variation (h = 0.249, HT = 0.305). Besides such features as relatively widedistribution, 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 varysignificantly with changing elevation, showing a trend that mid-elevation populations (2,200m and 2,600 m) were genetically more diverse than both low-elevation (1,800 m) andhigh-elevation populations (3,000 m and 3,400 m). H. rhamnoides subsp. sinensis is thoughtto be stressed by drought and high temperature at low elevations, and by low temperature athigh 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 middlealtitudinal zone, where favorable ecological conditions permit its continuous distributioncovering 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 andAFLPs followed the general pattern detected in woody species with widespread distributionsand outcrossing mating systems. Such plants possess more genetic diversity withinpopulations and less variation among populations than species with other combinations oftraits. 4. In the present study, Mantel tests showed positive correlations between altitudinaldistances and genetic distances among populations or subpopulations. The observedrelationship between altitude and genetic distances, and the result of the cluster analysisincluding populations or male subpopulations and classifying the groups into three altitudeclusters suggest that altitude is a major factor that restricts gene flow between populationsand subpopulations. 5. The analysis of molecular variance showed that only 3.8% of the variability residedbetween female and male subpopulations. Such a very restricted proportion of the totalmolecular variance between female and male subpopulations is due to common sexuality andmixing of genetic material between females and males. 6. The analysis based on ISSRs, AFLPs and RAPDs all revealed relatively high levelsof genetic variation among different altitudinal populations of H. rhamnoides subsp. sinensisin Wolong Natural Reserve of China. Their estimates of mean Nei’s gene diversity is equal to0.249, 0.214 and 0.170 respectively, suggesting the higher capacity of detecting geneticvariation of ISSR and AFLP than RAPD. It might be ascribed to their distinct sensitivity todifferent type of genetic variation. 7. Based on the coparative study on ISSR, AFLP and RAPD, we drew a conclusion thatthey all successfully reveal some useful information concerning the level and pattern ofgenetic vatiation occurring among different elevation populations of H. rhamnoides subsp.sinensis. AFLP is a ideal tool to taxonomic study and individual identification for theirhighest efficiency index and marker index among the three marker systems.

西藏齿突蟾蝌蚪冷适应策略研究

本文通过对高海拔两栖类西藏齿突蟾(Scutiger boulengeri)蝌蚪在实验室特定低温条件下的冷适应微空间行为分布的动态变化分析、温度耐受性实验及在不同适应温度的乳酸脱氢酶(LDH)同工酶的酶量与活性比较分析, 探讨了高海拔两栖类蝌蚪的部分冷适应策略。 西藏齿突蟾蝌蚪在不同温度的行为分布是一连续、动态过程，需用多种检验方法综合利用才能进行判断；在15℃, 除低海拔分布的西藏齿突蟾种群外所有实验物种蝌蚪均符合负二项分布、NeymanⅡ型分布；在10℃, 高海拔两栖类蝌蚪均符合负二项分布、NeymanⅡ型分布；在5℃、0℃低温时，高海拔两栖类不同分组的西藏齿突蟾蝌蚪的负二项分布、NeymanⅡ型分布均呈现明显差异, 这可能与高海拔两栖类蝌蚪在低温条件下通过不断地改变其行为分布方式来避免自身被冻伤有关。野外观察表明：高海拔两栖类蝌蚪常选择与流动河水相连的静水水体这种微生境中生存, 蝌蚪应对环境温度极端变化会不断改变其行为分布方式来选择最佳生存温度以避免极端高、低温对自身身体的伤害, 这种对微生境的利用能力对高海拔两栖类蝌蚪耐受极端环境温度的变化极其重要。 两栖类蝌蚪的温度耐受性实验表明不同的驯化温度可以改变西藏齿突蟾蝌蚪、两栖类仙琴水蛙蝌蚪的最适温度、逃避温度，并具有显著影响。 随着驯化温度5℃、10℃逐渐升高, 其最适温度、逃避温度也在一定范围内升高，但驯化温度对低海拔的仙琴水蛙蝌蚪的最适温度、逃避温度的改变效应大于高海拔的西藏齿突蟾蝌蚪的改变效应, 仙琴水蛙蝌蚪对温度的耐受范围、最适温度和逃避温度的ARRS值都大于西藏齿突蟾蝌蚪, 这说明仙琴水蛙蝌蚪对环境温度变化的适应能力大于西藏齿突蟾蝌蚪。 高海拔地区不同分组的两栖类蝌蚪, 在0℃适应温度时, LDH5条带的酶相对含量最高，而在5℃、10℃、15℃适应温度时，LDH5条带的酶相对含量明显都降低, 这表明酵解作用是高海拔两栖类蝌蚪的一些组织在低温﹑缺氧环境中的重要供能方式。高海拔两栖类蝌蚪同一分组的LDH总酶活性总是表现为10℃适应温度的总酶活性最高，而对低海拔的两栖类蝌蚪则是0℃适应温度的总酶活性最高, 这说明高海拔两栖类蝌蚪的LDH同工酶A、B两亚基基因活性在10℃时最高, 而低海拔两栖类蝌蚪的LDH同工酶A、B两亚基基因活性在0℃时最高。同时发现在15℃适应温度组的高海拔两栖类蝌蚪的LDH电泳图谱都有第6条带,有可能由LDH - C亚基组成, 对高海拔两栖类蝌蚪的LDH - C亚基只在15℃适应温度下才表达的机理还有待进一步的研究。 高海拔两栖类西藏齿突蟾蝌蚪通过行为分布方式的改变来选择最佳的生存温度, 这种温度选择过程与野外特定的微生境的存在密切相关, 现在由于人类对河道的不合理利用正在导致高海拔两栖类蝌蚪赖以生存的这种微生境逐渐消失, 这种微生境的消失将加速高海拔的两栖类种群数量衰退的进程。高海拔两栖类物种蝌蚪在低温（0℃）上表现出的同工酶多谱带说明，其A、B两亚基都有所表达，及其参与代谢的方式也是正常的，而低海拔两栖类物种蝌蚪只有A亚基表达的LDH5存在，因此其主要参与酵解过程，这种通过动物自身生理代谢方式的改变来适应极端环境温度条件的变化是高海拔两栖类蝌蚪能适应低温环境的重要策略。但高海拔物种的适应温度变化范围显著小于低海拔物种，对环境温度的变化适应能力有限，特别是对高温区域，因此全球气候变化可能对高海拔物种影响更为显著。 The partly cold-adaptation stratagem of the high altitude amphibian tadpole were researched in the laboratory by analyzing the high altitude amphibian tadpole of Scutiger boulengeri mainly on endpoints related to the dynamic variation of the micro-spatial behavior distribution patterns, the experiment of the temperature tolerance, and the enzyme content and activity of the lactic acid dehydrogenase(LDH) isozyme in special temperature condition. The behavior distribution of the Scutiger boulengeri tadpole is continuous and variable, but it can be figured out by multple testing ways. At 15℃, all of the experiment amphibian tadpoles behavior distribution fit both for the negative binomial distribution and NeymanⅡtype distribution except for the low altitude Scutiger boulengeri tadpoles. At 10℃, all of the high altitude amphibian tadpoles behavior distribution fit both for the negative binomial distribution and NeymanⅡtype distribution. At lower temperature, 5℃ and 0℃, the high altitude amphibian tadpoles of the Scutiger boulengeri at different groups behavior distribution fit for or don’t fit for behavior distribution respectively. It is denoted that the high altitude amphibian tadpoles probably avoid frostbiting by varying the behavior distribution patterns at low temperature condition. The high altitude amphibian tadpoles often actively select the special microhabitat which has the connected still water body and the flowing water body in the wild. It is important that tadpoles can endure the extreme temperature variety in this kind of microhabitat, because tadpoles can be better survival through select temperature condition through migrating in these kinds of microhabitats by varying their own behavior distribution patterns. Different acclimation temperature causes the significant change of preferred temperature(PT)、 avoiding temperature(AT) both in high altitude amphibian Scutiger boulengeri tadpoles and in low altitude amphibian Rana daunchina tadpoles in the temperature endurance experiment. With the acclimation temperature growing from 5℃ to 10℃. the PT and the AT of them would be uprise to some extent, but the effect of acclimation temperature on the PT and the AT of the tadpoles of Rana daunchina is more significant than the ones on the tadpoles of Scutiger boulengeri, at the same, the effects on the temperature endurance range, the ARRs of the tadpoles of Rana daunchina would be stronger than the ones on the tadpoles of Scutiger boulengeri. It is implied that the adaptation ability of tadpoles of Rana daunchina to the surroundings temperature alternation preferred to tadpoles of Scutiger boulengeri. At 0℃ acclimation temperature, the LDH5 enzyme comparative content of the high altitude amphibian tadpoles at different groups was highest, but it becomes lower at 5℃、10℃、15℃ acclimation temperature. It indicated that the alcoholysis role was the important ways of applying energy for special tissue of the high altitude amphibian tadpoles in low-temperature and low-oxygen condition. The total enzyme activity of the LDH of the high altitude amphibian tadpoles in the same group always keeps the highest at 10℃ acclimation temperature, but the low altitude amphibian tadpoles’ was maximum at 0℃. It was denoted that the gene activity of LDH -A and LDH – B submit was highest at 10℃ acclimation temperature for the high altitude amphibian tadpoles, but the low altitude amphibian tadpoles’ was maximum at 0℃. Meanwhile, the LDH electrophoretogram of the high altitude amphibian tadpoles always composed of 6 stripes at 15℃ acclimation temperature，the extra stripe probably was composed by LDH-C submit。It is unknown why LDH-C expresses only under high temperature。. The high altitude amphibian tadpoles can select the most optimal temperature by changing their behavior distribution patterns ceaselessly, but this course of selecting the most suitable temperature correlated with the special microhabitat in the wild closely. Nowadays, this kind of microhabitat which the high altitude amphibian tadpoles rely on are lossing gradually for human being exploit the riverway unreasonably. The disappearing of the microhabitat would accelerate the decline of the high altitude amphibian population. Compare to one band of LDH5, which only composed by the LDH-A submit, presents in the low altitude amphibian at 0℃, the five bands which composed by the LDH-A and LDH-B are checked out, this means the species which occurred in the highland is more adaptable to the low temperature. It is an important stratagem for the high altitude amphibian tadpoles adapt to the limited low temperature depends on the animal energy metabolism change.However, this kind of adaption is restricted, the adaption range to the temperature is much norrow in the high altitude amphibian than in the low one, especially for the high temperature side. The global climate change will be more serious for the high altitude species.

Are Chinese loess deposits essentially continuous?

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