Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H(Nei's genetic diversity) ranged from 0.11 to 0.20 and I(Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. > 1000 km), ISSR analysis is useful for determining genetic differentiations of C crispus populations including morphologically inseparable haploid and diploid individuals. (c) 2007 Elsevier B.V. All rights reserved.

Loss of allele diversity in introduced populations of the hermaphroditic bay scallop Argopecten irradians

The bay scallop Argopecten irradians is a hermaphroditic bivalve native to the Atlantic coast of the United States that was introduced to China for aquaculture production in 1982. It now supports a major aquaculture industry in China. Introduced species often start with limited genetic variability, which is problematic for the further selective breeding. Bay scallop aquaculture is exclusively hatchery based and as the initial introduction consisted of only 26 scallops, there have been concerns about inbreeding and inbreeding depression in cultured populations in China. In this study, eleven simple sequence repeat (SSR) markers were used to compare genetic variation in cultured populations from China with that in a natural population from the east coast of America. Although the difference in heterozygosity was small, the Chinese populations lost 9 of the 45 alleles (20%) found in the wild population. The reduced allele diversity suggests that the Chinese bay scallop populations experienced a bottleneck in genetic diversity that remains significant despite several recent introductions of new stocks aimed at expanding the gene pool. The loss of allele diversity may affect future efforts in selective breeding and domestication, and results of this study highlight the need for additional introductions, advanced breeding programs that minimize inbreeding and continued genetic monitoring. (c) 2007 Elsevier B.V. All rights reserved.

Genetic structure analysis of natural Sargassum muticum (Fucales, Phaeophyta) populations using RAPD and ISSR markers

Sargassum muticum is important in maintaining the structure and function of littoral ecosystems, and is used in aquaculture and alginate production, however, little is known about its population genetic attributes. In this study, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four populations of S. muticum and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China. The selected 24 RAPD primers and 19 ISSR primers amplified 164 loci and 122 loci, respectively. Estimates of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate level of genetic variations within each S. muticum population, and a high level of genetic differentiations were determined with pairwise unbiased genetic distance (D) and fixation index (F-ST ) among the populations. The Mantel test showed that two types of matrices of D and F-ST were highly correlated whether from RAPD (r = 0.9706, P = 0.009) or ISSR data (r = 0.9161, P = 0.009). Analysis of molecular variance (AMOVA) was conducted to apportion the variations among and within the S. muticum populations. It indicated that variations among populations were higher than those within populations, being 55.82% verse 44.18% by RAPD and 55.21% verse 44.79% by ISSR, respectively. Furthermore, the Mantel test suggested that genetic differentiations among populations were related to the geographical distances (r > 0.6), namely, conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. On the whole, the high genetic structuring among the four S. muticum populations along the distant locations was clearly indicated in RAPD and ISSR analyses (r > 0.9, P < 0.05) in our study.

Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei's unbiased genetic distance (D) and fixation index (F-ST) between the populations. The Mantel test showed that two types of matrices of D and FST were highly correlated, whether from RAPD or ISSR data, r=0.9310 (P = 0.008) and 0.9313 (P=0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.

Yellow perch strain evaluation I: Genetic variance of six broodstock populations

As a prelude to strain selection for domestication and future marker assisted selection, genetic variation revealed by microsatellite DNA was evaluated in yellow perch, Perca flavescens, from four wild North American populations collected in 2003-2004 (Maine, New York, North Carolina, and Pennsylvania,), and two captive populations (Michigan and Ohio). For the loci examined, levels of heterozygosity ranged from H-e=0.04 to 0.88, genetic differentiation was highly significant among all population pairs, and effective migration ranged from low (N(e)m=0.3) to high (N(e)m=4.5). Deviation from Hardy-Weinberg equilibrium was regularly observed indicating significant departures from random mating. Instantaneous measures of inbreeding within these populations ranged from near zero to moderate (median F=0.16) and overall inbreeding levels averaged F-IS=0.18. Estimates of genetic diversity, Phi(ST), and genetic distance were highest between Michigan and all other broodstock groups and lowest between New York and Ohio. Genetic differentiation among groups did not correlate with geographic distance. Overall, the patterns of variation exhibited by the captive (Michigan and Ohio) populations were similar to patterns exhibited by the other wild populations, indicating that spawning and management practices to date have not significantly reduced levels of genetic variation. (C) 2007 Elsevier B.V. All rights reserved.

Genetic variation in the endangered Anisodus tanguticus (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H-t and H-sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H-e and H-pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei's coefficient of differentiation (G(ST)) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.

Genetic variation and phylogeographic history of Picea likiangensis revealed by RAPD markers

Repeated cycles of retreat and recolonization during the Quaternary ice ages are thought to have greatly influenced current species distributions and their genetic diversity. It remains unclear how this climatic oscillation has affected the distribution of genetic diversity between populations of wind-pollinated conifers in the Qinghai-Tibetan region. In this study, we investigated the within-species genetic diversity and phylogenetic relationships of Picea likiangensis, a dominant forest species in this region using polymorphic DNA (RAPD) markers. Our results suggest that this species has high overall genetic diversity, with 85.42% of loci being polymorphic and an average expected heterozygosity (H (E)) of 0.239. However, there were relatively low levels of polymorphism at population levels and the differences between populations were not significant, with percentages of polymorphic bands (PPB) ranging from 46.88 to 69.76%, Nei's gene diversity (H (E)) from 0.179 to 0.289 and Shannon's indices (Hpop) from 0.267 to 0.421. In accordance with our proposed hypothesis, a high level of genetic differentiation among populations was detected based on Nei's genetic diversity (G (ST) = 0.256) and AMOVA analysis (Phi (st) = 0.236). Gene flow between populations was found to be limited (Nm = 1.4532) and far lower than reported for other conifer species with wide distribution ranges from other regions. No clusters corresponding to three morphological varieties found in the south, north and west, respectively, were detected in either UPGMA or PCO analyses. Our results suggest that this species may have had different refugia during the glacial stages in the southern region and that the northern variety may have multiple origins from these different refugia.

Patterns of genetic variation in Swertia przewalskii, an endangered endemic species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Phi(ST) comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory theta(B)) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.

Origin of mitochondrial DNA diversity of domestic yaks

Background: The domestication of plants and animals was extremely important anthropologically. Previous studies have revealed a general tendency for populations of livestock species to include deeply divergent maternal lineages, indicating that they were domesticated in multiple, independent events from genetically discrete wild populations. However, in water buffalo, there are suggestions that a similar deep maternal bifurcation may have originated from a single population. These hypotheses have rarely been rigorously tested because of a lack of sufficient wild samples. To investigate the origin of the domestic yak (Poephagus grunnies), we analyzed 637 bp of maternal inherited mtDNA from 13 wild yaks (including eight wild yaks from a small population in west Qinghai) and 250 domesticated yaks from major herding regions.Results: The domestic yak populations had two deeply divergent phylogenetic groups with a divergence time of > 100,000 yrs BP. We here show that haplotypes clustering with two deeply divergent maternal lineages in domesticated yaks occur in a single, small, wild population. This finding suggests that all domestic yaks are derived from a single wild gene pool. However, there is no clear correlation of the mtDNA phylogenetic clades and the 10 morphological types of sampled yaks indicating that the latter diversified recently. Relatively high diversity was found in Qinghai and Tibet around the current wild distribution, in accordance with previous suggestions that the earliest domestications occurred in this region. Conventional molecular clock estimation led to an unrealistic early dating of the start of the domestication. However, Bayesian estimation of the coalescence time allowing a relaxation of the mutation rateConclusion: The information gathered here and the previous studies of other animals show that the demographic histories of domestication of livestock species were highly diverse despite the common general feature of deeply divergent maternal lineages. The results further suggest that domestication of local wild prey ungulate animals was a common occurrence during the development of human civilization following the postglacial colonization in different locations of the world, including the high, arid Qinghai-Tibetan Plateau.

RAPD profiling in detecting genetic variation in endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA ( RAPD) markers were used to measure genetic diversity of Coelonema draboides ( Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P. R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% ( 161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.

银杉的遗传多样性及系统位置的研究一兼论松科的分子系统学

银杉属Cathaya是我国著名植物学家陈焕镛和匡可任建立的松科单型属，该属是否成立及该属在松科中的系统位置至今仍众说不一。银杉Cathazyaargyro phylla是银杉属的唯一幸存种，被誉为第三纪的活化石，并被列为我国一级保护的濒危植物，也是世界植物红皮书所列少数几种濒危裸子植物之一，保护区虽已设立数个，但该物种的濒危原因仍为未解之谜。本文针对上述问题开展了松科植物的分子系统学研究，探讨了银杉属的系统位置及松科的属间关系，并通过遗传多样性的研究探讨了银杉的濒危机制及保护策略。 1．松科的分子系统学研究 为选取合适的分子标记，本文先探讨了ITS-1在松科等裸子植物中的系统学价值。对1 6种裸子植物（其中含松科10属12种）的ITS-1的扩增结果如下：ITS-1在裸子植物属闻存在极大的长度变异(约600-2600bp)，因而不适用于属级以上的系统学研究;在同属种中，ITs-1的长度虽较一致，但与被子植物的ITS-1相比，裸子植物的ITS-1太长(如在松属中超过2600bp)，难以进行大规模研究。银杉属ITS-1的扩增产物中，一个片段的长度与松属ITS-1的长度相当，另一个片段的长度与云杉属ITS-l的长度相当，这三属间的关系值得进一步研究。此外，在松科植物ITS-1的研究中必须注意真菌“污染”这一问题。 由于ITS不适用于裸子植物属间关系的研究，因而本文运用rbcL-accD基因片段及trnK基因的PCR-RFLP分析探讨银杉属的系统位置及松科的属间关系。rbcL和trnK虽然都是叶绿体基因，但位于trnK内含子区的natK基因与rbcL基因在所受的选择压及进化速率上相差甚远，通过这两个基因研究结果的比较有助于评价系统树的可靠性。此外，考虑到松科的10个属中有9个属的rbcL基因序列已被测定，我们补测了银杉属的rbcL基因序列，进而对松科10个属的rbcL基因序列进行了全面分析。PCR -RFLP分析及rbcL基因的序列分析结果如下： A. PCR扩增的松科rbcL-accD基因片段均长约2550bp，属间没有明显的长度变异，1 8种限制性内切酶的单酶切及7种组合双酶切共获得86个酶切位点，其中54个为变异位点。PAUP和MEGA软件分析均表明：银杉属、松属、黄杉属和落叶松属构成一个单系群，且银杉属与松属的关系似乎近于它与另外二属的关系，但这一结果来得到bootstrap分析的较强支持;黄杉属近缘于落叶松属;冷杉属近缘于油杉属。 B．PCR扩增出的松科trnK基因均长约2557bp，在属间也没有明显的长度变异。16种限制性内切酶的单酶切及7种组合双酶切共获得92个酶切位点，其中68个为变异位点。变异位点中的35个具有系统发育信息，其中28个位于matK基因上。分布于整个trnK基因上的informative位点和仅分布于matK基因上的informative位点分别用于PAUP(version 3.1.1)分析，且PAUP分析中分别用Wagner简约法和Dollo筒约法构建系统树。总体看来，Wager简约树和Dono简约树的结构基本一致（Cedrus的位置除外）：Abies、Keteleeria、Tsuga和Pseudolarix分为一支，且这一支在所有的简约树中均得到分辨;Pseudotsuga近缘于Larix,而Abkes相对近缘于Keteleeria; Cathaya与Ables-Keteleeria-Pseudolarix-Tsuga这一支的关系较远，它与松科的其它属聚在一起，但b∞tstrap分析也不支持它与任何一个属有很近的关 系，说明Cathaya是比较孤立的一个属。Cedrus的位置较特殊，在Wagner简约树中它位于松属以外的其它8个属的基部（因松属为外类群），而在Dollo筒约树中它位于Al}ies-Kete~eer uz-Pseudo/zr/x- Tsuga达一支的基部，并且它与这一支的关系得到了bootstrap分析的较强支持。 C．银杉的rbeL基因长1425bp，其序列与松科其它9个属的rbcL基因序列均有较大差异，从遗传距离(Mega软件中的P-distance)上看，银杉属作为一个属是无可非议的。PAUp和Mega软件分析均表现：Abies、Keteleeria、Pseudolter和Tsuga分为一支，且Cathaya与这一支的关系较远;Pseuaiotsuga近缘于Larix,且这二属间的关系得到了bootstrap分析的强烈支持;Abies近缘于Keteleeria，Pseudolarix近缘于Tsuga，且这两支也得到了bootstrap分析的较强支持;Cathaya与Pinus聚为一支，但支持强度不高。Cetirus的位置与trnK基因的PCR-RFLP分析结果极为相似。 根据rbcL-accD基因片段及trnK基因的PCR-RFLP分析以及rbcL基因的序列分析可以得出如下结论：<1> Cathaya作为一个属不仅是成立的，而且是一个较为孤立的属，将它置于其它任何属内都是不正确的，该属与Abies-Keteleer ia-Pseudolarx-Tsuga这一支的关系较远，相对而言Cathaya与Pinus的关系可能稍近一些。<2>松科主要分为两大支：一支含Abies、KeteZeera、Pseudolarx和Tsuga，该支中Abies近缘于KetelaeriaPseudolarix近缘于Tsuga;另一支含Pseudotsuga、Larix、Cathaya、Pinus和Picete，该支中Pseudotsuga明显近缘于Larix;Cedrus可能更近于Abies-Keteleeria-Pseudolarix-Tsuga这一支。 2．银杉的遗传多样性研究 本研究运用随机扩增多态DNA(RAPD)方法对银杉的1 1 3个个体（分别采自现存全部4个居群：金佛山、花坪、大瑶山和八西山)进行了遗传多样性检测。21个1O-mer的寡核苷酸引物共检测11个位点，其中42个位点是多态的，占37%，这一多态位点百分率仅为裸子植物多态位点百分率平均值的一半左右，说明银杉的遗传多样性水平很低。对多态位点的分析发现银杉屠群内发生了十分严重的遗传漂变，因而导致屠群间的强烈分化。AMOVA分析表明：银杉的遗传变异中，34.7%的变异存在于居群闻，这一数值是裸子植物GST平均值(6。8%)的5倍多;在金佛山屠群中，17%的遗传变异存在于亚居群间，这一数值也是裸子植物GsT平均值的两倍多。银杉的这一居群遗传结构与其它裸子植物的居群遗传结构截然不同。此外，我们提出了度量遗传多样性水平的分化指数概念及其计算方法，并发现遗传多样性水平的高低与生境的复杂程度有一定的相关性。 银杉的遗传多样性水平很低，其适应幅必然很窄，且由于严重的遗传漂变导致居群间的强烈分化，使基因流受阻，进而产生严重的近交，近交又使银杉的适应能力进一步下降，并使极为有限的遗传多样性进一步丧失，这种恶性循环会导致银杉濒危程度的加剧，甚至绝灭。 鉴于银杉独特的居群遗传结构，即有相当大一部分遗传变异存在于居群之间，取样保护时，不仅要在每个居群中取足够多的个体，而且要在尽可能多的居群中取样。就原地保护而言，由于每个居群和亚居群都有独特的基因型，因而都具有保护价值。另外，人工加强居群间的基因流（如人工授粉）对提高银杉各居群的适应能力肯定是有益的。

三种兰科植物的保护遗传学研究

本研究在野外调查的基础上，采用随机扩增多态DNA (RAPD)分析和形态学方法，研究了我国三种珍稀濒危兰科植物硬叶兜兰（Paphiopedilummicranthum）、麻栗坡兜兰（P. malipoense）和独花兰（Changnienia amoena）的遗传多样性与群体遗传结构，主要结果如下： 1．采用1 2个引物对分布于我国云贵地区的4个硬叶兜兰群体共161个体进行RAPD扩增和分析，得出物种水平的多态条带百分率（PPB）为71.6%，Nci的基因多样度(h)为0.217，Shannon多样性指数（1)为0.3301;4个群体的平均多样性水平为PPB=45.2%，h=0.1457，1= 0.2204：低于远交兰花的平均水平。分子方差分析(AMOVA)表明，在总遗传变异中，群体间遗传变异占20.31%．群体内占79.69%;POPGENE给出的基因分化系数 （Gst）为0.2958;遗传分化略高于远交物种的平均水平。空间自相关分析表明，所检测的两个群体中存在明显的空间结构，基因型在群体中以不同的小斑块存在。遗传距离和空间距离不存在相关关系。 2．用于麻栗坡兜兰的RAPD引物同上，但取样范围只有贵州的2个群体共10个个体。就所研究的个体柬看，麻栗坡兜兰的遗传多样性明显低于远交兰花物种的平均水平。物种水平上，多态条带百分率(PPB)为49.5%。Nei的基因多样度（h）为0. 1174， Shannon多样性指数(I)为0.1764：在群体水平上，上述三个指标的平均值则分别为12. 75%、0.0486和0.0712，均大大低于硬叶兜兰。然而，尽管作了种种努力，麻栗坡兜兰的取样个体数量仍很少，因此所得结果可能会有误差。 3．用16个引物对分布于河南、湖北、湖南、江西4个省11个独花兰群体共216个体进行了RA PD扩增和分析，独花兰在物种水平PPB=80. 7%，h=0.197．1=0. 3116;在群体水平，上述三个指标的平均值则分别为40. 9%、0.1247和0. 1902，均低于远交兰花的平均水平。AMOVA分析表明，11个独花兰群体间的遗传变异占43.48%，群体内的占56.52%：在神农架和新宁地区内部，群体间的遗传变异分别占13.68%和49.3g%(AMOVA)。POPGENE给出的11个群体的基因分化系数(Gst)为0.3580．神农架和新宁地区内的Gst，值分别为0.1194和0.2597。可见，群体间的遗传分化明显高于远交物种的平均水平。空间自相关分析表明，独花兰的遗传变异在群体内不存在明显的空间结构。群体之间的遗传距离和空间距离不存在相关关系。 4．对独花兰7个群体形态性状的分析发现，12个形态性状在群体内均有较高的变异性，cv值变动于0.022-0.30O。庐山群体(LS)在所有性状上的平均值均为最高。营养性状和花部性状的变异性基本一致。除花葶长和花距直径与某些花部性状之间没有显著的相关关系外，各性状之间均有显著的相关性。对XN4群体的统计没有发现假磷茎数目与其他性状之间存在显著相关性。 根据以上对硬叶兜兰、麻粟坡兜兰和独花兰遗传多样性和群体遗传结构韵研究，结合其他方面的资料;对三种兰花的濒危机制进行了初步的分析。首先，人为采挖和破坏是导致这些兰花物种濒危的直接原因，尤其是麻栗坡兜兰。其次， 适宜兰花生存的生境正在只益萎缩、退化和片段化。这两方面因素的共同作用导致上述兰花群体的数目和规模日益下降，由此引发的遗传多样性降低和遗传结构的改变进一步加剧其濒危状况。对于独花兰而言，较低的繁殖能力又使其生存状态雪上加霜。针对三个物种不同的繁殖特性和遗传学状况，提出如下保护措施。(1)硬叶兜兰由于繁殖能力较强、现存个体尚多，遗传多样性损失不甚严重，因此以保护其所在的生境为基础、实施原位保护，是比较合适的保护策略。(2)麻粟坡兜兰目前受破坏程度非常严重;所剩个体很少，遗传多样性较低，已经很难进行有效的原位保护。因此;应利用迁地保护手段抢救目前尚存的个体。(3)独花兰的繁殖能力较弱，因此在保护生境和严禁采摘的基础上，可采用人工授粉等方式，提高结实率、增加繁殖效率，促使其复壮：在进行迁地保护时，则应注意不同群体间存在较大遗传变异而群体内多样性较低这一现实。

木根麦冬基因流动与居群遗传变异的时空动态研究

本报告对木根麦冬的生态、遗传与进化等方面进行了综合研究，分析了木根麦冬居群内个体育性变异与基因流的模式与强度，研究了木根麦冬居群遗传变异在时间与空间的动态变化过程，深入探讨了该物种的适应策略、自然选择与进化模式。同时对生物进化中的一些基本问题进行了尝试性探讨。 一、影响有性繁殖生物居群中交配与繁殖成功的行为、生态与遗传现象最终决定等位基因与基因型的相对频率及其在世代间的变化。植物的交配系统不仅决定了居群未来世代的基因型频率，而且还影响到植物居群的有效大小、基因流和选择等其它进化因素。我们利用等位酶多态性，采用似然性方法与计算机模拟为木根麦冬三个居群的种子确定父本。共有90.1%的种子可以在居群中找到唯一的最可能父本。在这些种子中，56.1%在0.95的显著水平上可以确定唯一亲本。并且还发现：1．个体自交越多，产生的种子就越多;2．个体提供 给自己的有效花粉比提供给别的植株的有效花粉多，3．只接受花粉而不提供花粉的植株产生的种子少;4．只接受花粉而不提供花粉的植株产生种子的机会也少，综合我们以前的研究结果，可以认为，木根麦冬的交配系统正在或已经发 生了转变，从以异交为主到以自交为主。 二、点模式分析的结果表明在所研究的三个居群中，木根麦冬个体都表现出相对随机的分布。空间自相关分析却发现开花个体在空间聚集分布，呈斑块状开花。这个结果也表明个体空间分布模式与开花状态没有直接关系，集团性开花可能是一个促进花粉传播的适应特征：开花个体的聚集可能对传粉者形成强有力的吸引。空间自相关分析表明木根麦冬居群中大多数等位基因都聚集成一定大小的斑块。基因的聚集突出的说明了木根麦冬居群内基因流动的状况，花粉与种子的传播受到限制。传粉能力弱的传粉者以及种子的近距离的散布使基因局限于一定的小范围内，从而形成基因斑块，而斑块的大小由传粉者的活动范围及种子的传播距离决定。 三、基因纯合导致有害的隐性基因或部分致死的基因表达，使纯合子适合度降低，因此纯合子频率在世代间的变化将是衡量近交衰退的一个较好的指标。木根麦冬中亲代居群的观察杂合度大大高于子代居群，这个结果表明大量的纯合子在从种子到成株的过程中被淘汰。大量的纯合子被淘汰可能时由于隐性致死或有害等位基因纯合的结果，自交以及有亲缘关系的个体之间交配使有害等位基因与致死等位基因纯合，从而使一个远交种中出现自交衰退，过量的纯合子被淘汰是自交衰退的直接后果。

无根萍生物学特性研究

无根萍属（Wolffia ）隶属于浮萍科天南星目，是世界上最小的被子植物。该属植物繁殖速度快;易于培养;结构简单，只具有一个雄蕊和一个雌蕊;自然状态下通常为克隆繁殖，遗传结构高度一致，具备特定研究目的模式植物的特点，正在或已经成为一些实验室研究光合作用、生物反应器、毒理学、生态修复和环境监测等的重要模式生物材料;同时还被作为建造航天生活仓和地外生命支撑系统的首选植物。该属植物蛋白含量高且氨基酸组分平衡，营养价值可与大豆相媲美。但该属植物一直是分类学界的疑难类群，不同的学者对该属的分类处理比较混乱;其次，对该属的生物地理研究也很不够，尤其是对国产类群的研究;另外，W. globosa 作为该属中国分布的物种，其生理学特性和形态结构发育还缺乏研究。为此，本文通过mat K 基因测序、RAPD 标记等手段，结合野外和室内的长期观测，对其分类和中国的地理分布以及生理学特性进行了研究。针对浮萍科植物作为水生植物，其对重金属和芳香烃衍生物的耐受逆境能力大小，和对淡水水体环境生态的指示作用。本文研究了W. globosa 具解毒功能的谷胱甘肽转硫酶的活性;最后，探索了从黄鳝（Monpterus albus Zuiew ）中分离纯化GSTs 的技术与方法并对maGST 的部分特性进行了研究。主要研究结果如下： 1. Wolffia 系统分类学研究 前人认为，Wolffia 柱头的颜色是重要的分组、分种检索性状。我们对其长期、活体、原位、实时跟踪观测结果表明，柱头颜色是Wolffia 个体发育上的变化过程，不是一个稳定性状，用作Wolffia subgroup 内组的划分特征和种的鉴别特征是不适合的。在此基础上我们重新修定了该属的分种检索表。利用形态分类学性状——气孔、长/宽、高/宽以及最大宽度在水面上还是水面下等性状，认为中国分布的类群应是W. globosa，但亦有W. neglecta 存在的证据。mat K 基因片段结果支持形态学的结论。通过广泛的野外采集，在我国北京、河北和吉林发现Wolffia 的新分布。 2. 中国Wolffia 居群遗传学研究 以RAPD 分子标记对广泛分布的居群遗传多样性研究表明，无根萍属植物主要以无性繁殖方式繁育，居群主要由单一克隆后代组成，如海河流域以及松花江流域居群;但一些居群亦兼有性繁殖方式，并具较高的遗传多样性，如武汉、海南居群。利用MVSP, Popgene 和Ntsys 等分析方法探讨了中国产Wolffia 居群遗传多样性和地理分布格局间关系。 3. W. globosa 的生理学研究 建立了较为完善的W. globosa 的无菌培养和保存体系。W. globosa 在逆境中，会形成休眠体;同时，发现不同居群甚至不同克隆系之间其抗逆性和生长速度存在着显著差异，差异最大的如海南文昌居群的生长速率，是长春居群的4.19 倍;不同的时间统计生长周期存在着不同结果，生长节律每天有两个生长高峰呈双“S”型;W. globosa 的耐受温度范围和pH 范围广;低浓度的IAA，GA，6-BA， EDDHA-Fe 以及EDTA 等物质具有促进W. globosa 生长的特性;但是，所有这些处理均没能促使W. globosa 从营养生长转入生殖生长。 4. W. globosa 的解剖学研究 W. globosa 通常是进行克隆繁殖，通过组织切片发现无性分枝子体还未伸出母株之前就已经完成分化，与此同时分枝子体中又分化出新的子体，分枝呈聚伞状，子体生长方向彼此相对;另外，生殖生长结构的分化也是在母体中完成的;生殖生长点与营养生长点不是同一生长点。 5. 浮萍科植物的毒理学研究 以重金属Cr3+和芳香烃衍生物CDNB 溶液处理Wolffia，Spirodela 和Lemna， 三种水生生物，结果表明Wolffia 比Spirodela， Lemna 对重金属和芳香烃衍生物有更强的抗逆能力，如在同等条件下对于Cr3+Wolffia 的半致死剂量800GB(≈ 80mg/L)，而Spirodela 和Lemna 则分别为10mg/L;20mg/L;表明W. globosa 是一个优良的生态环境修复植物。与此同时，研究了W. globosa 中具有解毒功能的GSTs 粗酶液活力在不同浓度的重金属离子（Cu2+和Cd2+）以及芳香烃衍生物（CDNB 和NBD-Cl）随处理时间的变化情况。 6. 从水生生物黄鳝Monpterus albus Zuiew 中分离纯化GSTs 的研究GSTs 活性的变化是环境监测的一个Biomarker，为此研究从M. albus 中分离 纯化GSTs 的技术与方法。经GSH 亲和层析纯化的酶活力为粗酶液的207 倍，进而鉴定了maGST 的部分特性。SDS-PAGE 电泳和MALDI-TOF/MS 表明MaGST 为同源二聚体，分子量约为52kDa，单亚基分子量约为26 kDa。maGST 酶动力学表明对CDNB 为13.07 ± 0.37 微摩尔每分钟每毫克蛋白;对NBD-Cl 为5.54 ± 微摩尔每分钟每毫克蛋白;对ECA、4-NPA 几乎没有活性。在GSH 底物饱和，CDNB 的Km 值和Vmax 分别为0.32 mM 和16.19 微摩尔每分钟每毫克蛋白;CDNB 底物饱和，GSH 的Km 值和Vmax 分别为0.44 mM 和28.83 微摩尔每分钟每毫克蛋白。maGST 的酶活性pH 值较宽，温度范围广：在pH7.0-7.5 具有最大速度，在pH6.5 和pH8.5 时分别具有65%和72%的酶活力;在45℃时具有最大活性，30℃和55℃时为最大活力的80%，60℃几乎完全丧失酶活力。

稻属Oryza rufipogon和Oryza nivara的遗传多样性和居群分化研究

普通野生稻(Oryza rufipogon Griff.)和尼瓦拉野生稻(O. nivara Sharma et Shastry)是亚洲栽培稻的最近缘野生种，是亚洲栽培稻育种和遗传改良的重要基因库。由于人类的破坏、生境片段化及亚洲栽培稻的遗传渐渗等因素，这两种野生稻处于濒危状态而急需保护。本文用微卫星标记研究中国普通野生稻居群的遗传多样性和居群分化，用核基因序列研究了全分布区的普通野生稻和尼瓦拉野生稻的遗传变异及模式，同时发现了巴布亚新几内亚普通野生稻的特殊分子变异。主要结果如下： 取自中国普通野生稻四个主要分布区的12个天然居群，共237份个体材料，利用10对微卫星引物对遗传多样性进行比较研究。结果表明这些居群的遗传多样性处于中高水平，其中每个位点的等位基因数之在2至18之间不等，平均10.6个，多态位点比率P从最低40.0%最高100%，平均为83.3%。观察杂合度在 (HO) 0.163-0.550变化，平均为0.332, 期望杂合度(HE) 在0.164- 0.648，平均 0.413，从地区来看，广西居群的遗传多样性水平是最高的，其次是广东、海南居群，江西的最低。这些结果与以前用其他分子标记得到的结果基本一致。但是本研究得到的居群间遗传分化却相对较高，分化系数RST为 0.5199，θ为 0.491，这表明一半左右的遗传变异存在于居群之间。两两居群之间的分化系数(pairwise θ)与地理距离呈正相关(r = 0.464)，这表明中国普通野生稻居群之间的遗传分化是由于地理的隔离造成的。造成目前这种遗传结构和分化模式的主要原因在于普通野生稻生境的破坏和生境的片段化。 选择6个普通野生稻居群，5个尼瓦拉野生稻居群共11个居群105个个体材料为研究对象，取样覆盖两个野生稻的主要分布区，用核基因片段Lhs1研究了这两种野生稻的序列水平的变异，探讨了它们的遗传结构和基因流方向。结果表明，普通野生稻的核苷酸多样性，无论在居群还是物种水平上，均显著高于尼瓦拉野生稻的。在居群水平上，普通野生稻的核苷酸多样性(Hd = 0.712; θsil = 0.0017)是尼瓦拉野生稻的2-3倍(Hd = 0.306; θsil = 0.0005)。AMOVA的结果表明，尼瓦拉野生稻居群之间的遗传分化(78.2%)显著高于普通野生稻居群间的分化(52.3%)。造成这种不同层次的遗传多样性和完全不同的遗传分化模式的原因在于这两种野生稻差异显著的生活史和交配系统。普通野生稻是多年生、异交的野生种，营养与有性繁殖混合，尼瓦拉野生稻为一年生的自交种，靠种子繁殖。模拟分析表明，二者之间的基因流方向是单向的，明显是从自交的尼瓦拉野生稻到异交的普通野生稻，基因流在这两个野生 稻遗传多样性模式的形成上，扮演至关重要的角色。 对于稻属A基因组物种的进化和多样性来说, 巴布亚新几内亚(PNG)是个很重要的地方。我们用两个核基因和一个叶绿体基因，对巴布亚新几内亚的普通野生稻样品进行了序列分析，结果发现来自巴布亚新几内亚北部Sepik河谷的普通野生稻样品在核基因Os0053位点和叶绿体基因序列上与其他地区的样品有很大的差异。巴布亚新几内亚北部的普通野生稻在Os0053位点上杂合的，其中一条等位基因与普通野生稻基本一致，另一条等位基因则与A基因组的另一个物种O. meridionalis基本一致，证明巴布亚新几内亚北部的普通野生稻实际上是亚洲典型普通野生稻与澳洲野生稻O. meridionalis种间进行天然渐渗杂交的后代。这个结果从分子序列上验证了前人有关巴布亚新几内亚北部的普通野生稻是比较特殊的观点。