准噶尔无叶豆(Eremosparton songoricum(Litv.)Vass.)是豆科无叶豆属小半灌木，既能开花结实进行有性繁殖，又可以靠根茎进行无性克隆繁殖，为稀有的国家三级保护植物，在我国仅片段化分布于新疆古尔班通古特沙漠局部区域。本文结合种群繁育生物学的部分研究结果，采用ISSR分子标记对采自古尔班通古特沙漠腹地及边缘的7个准噶尔无叶豆自然居群共148个个体进行了遗传变异和克隆多样性分析，探讨了居群遗传分化程度及其成因，同时为制定适宜的保育策略提供基础数据。研究结果表明： (1)分别用2×CTAB法、NaOH法及直接PCR扩增法三种方法制备PCR模板，比较扩增谱带，结果表明三种方法均能产生稳定、可靠、重复性好的PCR扩增谱带。其中，2×CTAB法提取的DNA产量及质量较高，PCR扩增谱带稳定、清晰，但该方法操作步骤多，单个样品处理时间长，耗时耗力；NaOH法制备模板DNA简单快速，经济高效，在1－2h可提取10－20个样品DNA，比常规方法快3－5倍，并可产生稳定、可靠、重复性好的PCR扩增谱带；而直接扩增法是一种更加快速、简便、廉价的制备方法，符合快速节省的原则，适宜制备大批量的PCR模板。研究结果对其它荒漠植物，尤其是叶片极端退化或珍稀濒危植物的PCR模板制备具有具体的指导意义。 (2)8个引物共扩增了84个位点，其中77个为多态性位点，物种水平上的多态位点百分比PPB为91.67%，Nei's基因多样性指数I为0.3192，Shannon信息指数H为0.3540；居群水平上的多态位点百分比PPB为58.45%，Nei's基因多样性指数I为0.2248，Shannon信息指数H为0.3270，和其它荒漠植物相比，准噶尔无叶豆表现出较高的遗传变异水平，说明准噶尔无叶豆是具有较高遗传多样性的狭域分布特有种。这可能是因为准噶尔无叶豆有性兼无性生殖的繁育方式可以使种群维持较高水平的遗传变异。 (3)本研究得到的居群间总的分化系数GST为0.2978，采用AMOVA进一步分析发现，有31.88%的遗传变异存在于居群间，表明居群间存在显著的遗传分化。①居群间较高水平的遗传分化表明居群间的基因流是很有限的，准噶尔无叶豆生境片段化而导致的基因交流障碍是造成居群间遗传分化最主要的原因；②从准噶尔无叶豆群落可划分为5大群丛并表现出不同的群落特征，说明不同微生境所造成的环境压力可能会对不同基因型进行选择，导致居群之间的遗传分化。 (4)克隆多样性分析表明，居群水平上G居群的Simpson多样性指数和均匀度指数最高，分别为0.9400和0.9885，E居群最低，分别为0.8457和0.9021；物种水平上，Simpson多样性指数为0.9858，分布均匀度指数为0.9673。准噶尔无叶豆所有居群均为多克隆居群，局部基因型所占比例达到93.92%，没有广布基因型，说明准噶尔无叶豆基因型的地域性分布强。认为克隆繁殖的自然属性以及片断化的居群分布格局对克隆基因型分布产生了影响，此外，推测准噶尔无叶豆居群在建立之初可能是由多个不同基因型个体组成，导致了丰富的克隆多样性。 (5) 基于以上研究结果，从遗传学角度考虑，对于该物种的保护应从以下几个方面开展：①保护准噶尔无叶豆的栖息地，减少人为干扰和破坏。②保护准噶尔无叶豆的遗传完整性。在取样策略上，由于居群间有明显的遗传分化，因此，要广泛采集尽可能多的居群的种子。③尽管准噶尔无叶豆群体中平均克隆所含分株数不多(每个基株平均只有1.05－2.27个分株)，但不同克隆间相差很大，意味着不同克隆可能具有不同的生长潜力或适合度，从而可能具有不同的竞争能力。因此，有必要加强对不同克隆系的保护力度，在采集种子时要尽可能采集不同克隆系的种子。Eremosparton songoricum(Litv.)Vass. is a clonal shrub that reproduces both asexually by below-ground rhizomes and sexually by seeds. Having been severely disturbed by human activities, it has greatly declined in range, population number and population size over the past decades. It is now a rare species with a narrow distribution in fragmented habitat patches in the Gurbantunggut Desert of Xinjiang, China. Inter-simple sequence repeats (ISSR) were used to evaluate the genetic variation and clonal diversity of seven populations (148 individuals) of E. songoricum from Gurbantunggut Desert. The main results are highlighted as follows: (1)Three methods preparing DNA template for PCR amplification, 2×CTAB, NaOH and direct PCR amplification. The results showed that the genome DNA obtained from three methods could meet for ISSR reaction. Among which, direct PCR amplification was the most rapid, simple and lowest cost method, but the stability of PCR banding patterns was influenced by the amount of plant material. Once the total amount of material was controlled and the PCR amplification system was built, the method was considered to be best for a large-scale PCR test. (2)A total of 84 discernible DNA bands were generated using eight primers of which 77 were polymorphic, indicating considerable genetic variation at the species level (PPB = 91.67%, I = 0.3192) and species level (PPB = 58.45%, I =0.2248) compare to other desert plants. The factors leading to the high genetic variation of the species were analyzed. First, it is probably due to outcrossing or mixed-mating breeding system. Second, the somatic mutation may play an important role in the genetic variation of higher plants, especially in groups which lacking of sexual reproduction. (3)Clonal diversity was also high with Simpson diversity index (D) varied from 0.8457 to 0.9400 at population level and 0.9858 at species level. The evenness of distribution of genotypes in E. songoricum populations is 0.9673 at species level, with all of the genotypes being local ones. This may indicate that multi-clone origin of populations, and clonal reproduction also has an important influence on the genetic structure of E. songoricum populations. (4)A relatively high value of GST(0.2978) indicate significant genetic differentiation among populations. Analysis of molecular variance(AMOVA) further revealed that 31.88% of ISSR variation resided among populations(p<0.341). Barriers of gene flow caused by habitat fragmentation and population size reduction may contribute to the significant genetic differentiation among populations. Additionally, the environmental pressure of microhabitat may caused genetic differentiation between populations, suggesting that selection-favouring adaptations to local environments have played an role in determining the current genetic profile of this species. (5)The results provide baseline data for formulating conservation strategies for this species. Conservation strategies should include both in situ and ex situ approaches to preserve the greatest possible extent of the genetic diversity of the species
