两种濒危水韭植物迁地保护居群的基因流动态及回归重建保育遗传管理策略

采用微卫星分子标记对中华水韭（Isoetessinensis）安徽休宁、浙江建德和东方水韭（I.orientalis）浙江松阳三个孑遗居群的迁地保护居群开展了遗传多样性检测与遗传结构分析。7对多态性微卫星引物在36个迁地保护亚居群的720个样本中共检测到59个等位基因,每位点平均等位基因数（A）为8·43。迁地保护亚居群均维持很高的遗传多样性,多态信息含量（PIC）平均为0·707。迁地保护亚居群间遗传分化较低,遗传分化系数GST仅为0·070,居群间具有较大基因流（Nm=3·59）。单因素方差分析发现水韭孢子或孢子体在沿主要水流方向上的长距离传播能力要强于弱水流方向上的短距离传播能力,水流动态对水韭植物的基因流有重要影响。这与UPGMA聚类分析中迁地保护亚居群按邻近位置或水流相通程度优先聚类的结果相一致,水流所带动的强大基因流导致了不同孑遗居群来源的迁地保护亚居群间的遗传混杂。建议在开展水韭植物的迁地保护或回归自然重建时,对具有地方适应分化或者显著性进化的水韭植物居群应相互隔离而不宜配置在一起,以避免远交衰退的遗传风险。     

我国盾叶薯蓣居群遗传结构分析

利用随机扩增多态DNA(RAPD)标记,分析研究了中国11个盾叶薯蓣居群82个个体的遗传多样性与遗传结构,15个寡聚核苷酸引物扩增共得到108条带,其中96条为多态带,占88．89％。Shannon多样性指数(I)为0．3093,居群水平的变异从0．1564到0．3098,物种水平的Nei基因多样度(h)为0．2499,居群水平的变化范围为0．1607到0．2137。遗传变异分析表明,物种水平的基因分化系数Gst为0．3415,居群间的基因流Nm为0．9641,居群间遗传交换小。分子方差分析(AMOVA)表明,居群内变异为68．96％,地区间变异为19．45％,居群问变异为11．58％。聚类结果以长江为界,将盾叶薯蓣分为南北两个大类群。研究结果对盾叶薯蓣种质的迁地保护有重要意义。     

Assessment of the domestication state of ackee (Blighia sapida K.D. Koenig) in Benin based on AFLP and microsatellite markers

Ackee (Blighia sapida) is a native multipurpose species important for the livelihoods of the rural populations in Benin. Trees are found in natural forests or are managed by farmers in different traditional agroforestry systems. Genetic variation at amplified fragment length polymorphism (AFLP) markers, four nuclear microsatellites (nSSRs) and one chloroplast microsatellite (cpSSR) were investigated in 279 individuals from six wild and eight cultivated populations from Benin. The AFLP data revealed moderate levels of diversity of ackee in Benin (mean diversity values are proportion of polymorphic loci = 52.8% and Nei??s gene diversity = 0.157, for 375 AFLP fragments). The mean diversity values based on nSSR-markers are expected heterozygosity = 0.286, allelic richness = 2.77. Genetic variation of wild and cultivated populations did not differ markedly. AMOVA revealed that only 7.3 and 5.2% of the variation was partitioned among populations for nSSR- and AFLP-markers, respectively. A Mantel test based on these both marker-types revealed significant correlations between population pairwise geographic distance and genetic differentiation. Differentiation among cultivated populations was higher than among wild populations. The only polymorphic chloroplast microsatellite marker (ccmp7) showed three haplotypes. Cultivated populations from northeastern Benin were fixed on one haplotype which was not observed elsewhere indicating a different origin of these populations possibly from neighboring Nigeria. Farmer-led domestication had an impact on the spatial distribution of genetic variation but did not result in significant losses of diversity within populations. Measures to conserve genetic resources of ackee in each of the three main bioclimatic zones in Benin are proposed.     

大王马先蒿云南五个居群遗传变异研究

采用随机扩增多态DNA(RAPD)分子标记方法,对大王马先蒿(PedicularisrexL.)分布在云南中甸、丽江、大理、武定和昆明的5个居群(其中中甸居群有3个亚居群)的遗传多样性和遗传结构进行了研究。结果表明,大王马先蒿居群具有较高的遗传多样性,多态位点百分率P、Shannon表型多样性指数I和Nei的基因多样性指数h分别为82.0%、0.361和0.240,遗传多样性水平与居群的地理分布范围相关;大王马先蒿居群间的遗传变异较高,遗传变异主要发生在居群之间,而不是居群内部;居群间遗传分化系数Gst为0.747,遗传分化明显,居群间基因交流较少。居群间的遗传距离与居群间的相对地理距离具有一定的相关性。对云南西北部中甸居群的3个亚居群的遗传多样性分析表明,居群的遗传多样性水平与海拔高度有一定的相关性。居群间遗传变异较高可能是由于大王马先蒿为短命多年生植物,繁育系统为混交型,且自交占较大比例等原因造成的。     

Genetic variation and conservation assessment of Chinese populations of Magnolia cathcartii (Magnoliaceae), a rare evergreen tree from the South-Central China hotspot in the Eastern Himalayas

Nine natural populations of the rare evergreen tree Magnolia cathcartii (Magnoliaceae) were sampled across its natural range, and amplified fragment length polymorphism (AFLP) markers were used to assess genetic variation within and among populations. Three ex situ populations were also surveyed to determine whether conservation plantings include the entire genetic diversity of the species. Genetic diversity within the natural populations was very low (0.122 for Nei’s gene diversity), and the southeast populations had the highest diversity. The ex situ populations had a lower diversity than the mean diversity for all populations, and none of the ex situ populations reached the levels of diversity found in their source populations. Genetic differentiation was high among natural populations (G _st = 0.247), and an isolation-by-distance pattern was detected. Habitat fragmentation, restricted gene flow, and geitonogamy are proposed to be the primary reasons for the low genetic diversity and high genetic differentiation. More protection is needed, especially for the southeast populations, which possess the highest numbers of unique alleles according to AFLP fragment analyses. The ex situ program was a good first step towards preserving this species, but the current ex situ populations preserve only a limited portion of its genetic diversity. Future ex situ efforts should focus on enhancing the plantings with individuals from southeastern Yunnan.     

Genetic diversity and differentiation in Eryngium alpinum L. (Apiaceae): comparison of AFLP and microsatellite markers

Genetic diversity and structure of 12 populations of Eryngium alpinum L. were investigated using 63 dominant amplified fragment length polymorphism (AFLP) and seven codominant microsatellite (48 alleles) markers. Within-population diversity estimates obtained with both markers were not correlated, but the microsatellite-based fixation index Fis was correlated with both AFLP diversity indices (number of polymorphic bands and Nei's expected heterozygosity). Only AFLP diversity indices increased with the size of populations, although they did not significantly differ among them (Kruskall-Wallis test). The discrepancy between AFLPs and microsatellites may be explained by a better coverage of the genome with numerous AFLPs, the higher mutation rates of microsatellites or the absence of significant difference among within-population diversity estimates. Genetic differentiation was higher with AFLPs (theta=0.40) than with microsatellites (theta=0.23), probably due to the higher polymorphism of microsatellites. Thus, we considered global qualitative patterns rather than absolute estimates to compare the performance of both types of markers. On a large geographic scale, the Mantel test and multivariate analysis showed that genetic patterns were more congruent with the spatial arrangement of populations when inferred from microsatellites than from AFLPs, suggesting higher homoplasy of AFLP markers. On a small spatial scale, AFLPs managed to discriminate individuals from neighboring populations whereas microsatellites did not (multivariate analysis), and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with microsatellites. However, dominant AFLPs cannot be used to study heterozygosity-related topics. Thus, distinct molecular markers should be used depending on the biological question and the geographical scale investigated.     

Genetic diversity of Annona crassiflora (Annonaceae) in northern Minas Gerais State

Genetic diversity analyses of tropical tree species are relevant to landscape management, plant genetic resource inventory, and biological conservation of threatened species. Annona crassiflora is an endangered fruit tree native to the Cerrado biome that is threatened by reduction of natural populations and fruit extraction. We examined the intra- and interpopulational genetic diversity of this species in the northern region of Minas Gerais State. Seventy-two individuals from four natural populations were genotyped using RAPD markers. We found moderate genetic diversity among populations, with Shannon's I index varying between 0.31 and 0.44, and Nei's genetic diversity (H(E)) for the population set equal to 0.31. AMOVA indicated a greater genetic variation within (77.38%) rather than among populations (22.62%), tending towards isolation by distance (Mantel's r = 0.914; P = 0.089). Nei's genetic identity estimates among populations revealed a hierarchical pattern of genetic similarity of form [(CA1, CA2), MC], [(GM)], corroborating the high genetic differentiation between spatially isolated populations.     

A study of conservation genetics in Cupressus chengiana, an endangered endemic of China, using ISSR markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (He) was 0.3120, effective number of alleles (Ae) was 1.5236, and Shannon's information index (I) was 0.4740. At the population level, PPB = 48%, Ae = 1.2774, He = 0.1631, and I = 0.2452. Genetic differentiation (Gst) detected by Nei's genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (Nm) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r = 0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.     

栲树天然群体遗传结构的RAPD分析

利用RAPD分子标记对 5个栲树 (CastanopsisfargesiiFranch .)天然群体共计 188个个体的遗传多样性和群体遗传结构进行了分析。 4 1个随机寡核苷酸引物共检测到 385个位点 ,其中多态位点 15 7个 ,占 4 0 .78%。物种水平的Shannon多样性指数I=0 .4 5 97,Nei基因多样度h =0 .2 96。遗传变异分析表明 ,栲树群体的遗传变异主要存在于群体内 ,利用Shannon多样性指数估算的分化 (Hsp_Hpop) /Hsp=0 .0 4 76 ,遗传分化系数Gst =0 .0 4 2 9,分子方差分析 (AMOVA)也证实了这一结论 ,群体内的变异组分占了 94 .97% ,群体间变异只占 5 .0 3%。AMOVA分析结果的显著性检验也表明 ,群体间及群体内个体间均呈现出显著分化 (P <0 .0 0 1)。     

Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88.5%) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84.8%), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on phiST was moderate (Nm=1.395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species.     

Multilocus population structure of Tapesia yallundae in Washington State

Population genetic structure of the fungal wheat pathogen Tapesia yallundae in Washington State was determined using genetically characterized amplified fragment length polymorphic (AFLP) markers and mating-type (MAT1-1 or MAT1-2). Segregation and linkage relationships among 164 AFLP markers and MAT were analysed using 59 progeny derived from an in vitro cross. Alleles at 158 AFLP loci and the mating-type locus segregated in a 1:1 ratio. Ten unlinked markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation among five subpopulations of T. yallundae representative of the geographical distribution of wheat production in eastern Washington. Among 228 isolates collected, overall gene diversity was high (h = 0.425) and a total of 91 unique multilocus genotypes (MLG) were identified, with 32 MLG occurring at least twice. The overall population genetic structure was consistent with random mating based on the segregation of mating-type, index of association (IA), parsimony tree length permutation test (PTLPT) and genotypic diversity analyses. However, clonal genotypes were found within each subpopulation and were also distributed among the five subpopulations. No significant differences in allele frequencies were found among the five subpopulations for all 10 loci based on contingency table analysis (G2) and Wier & Cockerham's population differentiation statistic theta (theta = -0.008, P = 0.722). T. yallundae appears to consist of a large homogeneous population throughout eastern Washington with both sexual and asexual reproduction contributing to the observed population genetic structure despite no report of sexual fruiting bodies of T. yallundae occurring under natural field conditions.     

濒危平胸龟两个自然居群的ISSR分析

利用22个ISSR引物研究了28个来自广东从化居群和21个来自福建寿宁居群的野生平胸龟(Platysternon megacephalum)的遗传多样性。结果表明,分布于这两个地区的平胸龟总居群和居群内均具有较高的遗传多样性,而广东从化居群遗传多样性高于福建寿宁居群。居群间的遗传变异度FST=0.3715(P<0.001),遗传分化系数Gst为0.2059。NJ树聚类分析显示,广东从化居群和福建寿宁居群各自聚成两个类群,有较明显的居群分化,具有一定的地理区域性,但基因流Nm为1.9279,这表明平胸龟两自然居群间存在一定基因流。此外,结合BSA技术,从11条引物中筛选出11个可作为鉴别广东从化居群或福建寿宁居群的分子标记。     

Genetic Diversity of Phaeoisariopsis griseola in Kenya as Revealed by AFLP and Group-specific Primers

Genetic diversity of 50 Phaeoisariopsis griseola isolates collected from different agroecological zones in Kenya was studied using group‐specific primers and amplified fragment length polymorphism (AFLP) markers. Group‐specific primers differentiated the isolates into Andean and Mesoamerican groups, corresponding to the two common‐bean gene pools. Significant polymorphisms were observed with all the AFLP primer combinations used, reflecting a wide genetic diversity in the P. griseola population. A total of 207 fingerprints was generated, of which 178 were polymorphic. Cluster analysis of the polymorphic bands also separated the isolates into the two groups defined by group‐specific primers. All the isolates examined were grouped into three virulence populations; Andean, Afro‐Andean and Mesoamerican, and their genetic diversity measured. On average, greater diversity (91%) was detected within populations than between populations (9%). The genetic distance between Andean and Mesoamerican populations was higher (D = 0.0269) than between Andean and Afro‐Andean (D = 0.0095). The wide genetic diversity reported here has significant implications in breeding for resistance to angular leaf spot and should be taken into consideration when screening and deploying resistant bean genotypes.     

Nuclear DNA microsatellite analysis of genetic diversity and gene flow in the Scandinavian brown bear (Ursus arctos)

In the 1930s, the Scandinavian brown bear was close to extinction due to vigorous extermination programmes in Norway and Sweden. Increased protection of the brown bear in Scandinavia has resulted in the recovery of four subpopulations, which currently contain close to 1000 individuals. Effective conservation and management of the Scandinavian brown bear requires knowledge of the current levels of genetic diversity and gene flow among the four subpopulations. Earlier studies of mitochondrial DNA (mtDNA) diversity revealed extremely low levels of genetic variation, and population structure that grouped the three northern subpopulations in one genetic clade and the southernmost subpopulation in a second highly divergent clade. In this study, we extended the analysis of genetic diversity and gene flow in the Scandinavian brown bear using data from 19 nuclear DNA microsatellite loci. Results from the nuclear loci were strikingly different than the mtDNA results. Genetic diversity levels in the four subpopulations were equivalent to diversity levels in nonbottlenecked populations from North America, and significantly higher than levels in other bottlenecked and isolated brown bear populations. Gene flow levels between subpopulations ranged from low to moderate and were correlated with geographical distance. The substantial difference in results obtained using mtDNA and nuclear DNA markers stresses the importance of collecting data from both types of genetic markers before interpreting data and making recommendations for the conservation and management of natural populations. Based on the results from the mtDNA and nuclear DNA data sets, we propose one evolutionarily significant unit and four management units for the brown bear in Scandinavia.     

Original birthplace of cultivated common buckwheat inferred from genetic relationships among cultivated populations and natural populations of wild common buckwheat revealed by AFLP analysis

The genetic relationships among seven cultivated populations and eight natural populations of wild common buckwheat were analyzed using amplified fragment length polymorphism (AFLP). The genetic distance was estimated for each pair of the 15 populations based on the AFLP data, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the genetic distance. All the cultivated populations were grouped in a cluster. The natural populations were grouped into two clusters composed of (1) the Sanjiang group (three populations from eastern Tibet and one population from Adong village of Yunnan province) and (2) two populations from Yunnan province and two populations from Sichuan province. The Sanjiang group is more closely related to cultivated populations. These results indicate that the direct ancestor of common buckwheat was natural populations of wild common buckwheat from the Sanjiang area.     

Genetic Variation in the Endangered Endemic Species Cycas fairylakea (Cycadaceae) in China and Implications for Conservation

Cycas fairylakea is an endangered endemic species in China. Genetic diversity within and among four natural populations of this species in China was investigated using amplified fragments length polymorphism (AFLP). A moderate to low level of intraspecific genetic diversity was detected in this species (at population level: P = 39.57 %, H₀ = 0.244; at species level: P = 60.22%, H₀ = 0.356). The among-population component accounted for, respectively, 25.7 and 31.5% of the genetic variation, according to AMOVA and Shannon’s index, indicating most of the genetic variation was found between individuals within populations. All four populations have opposite pyramid age structure, and few coning individuals, which is still decreasing. Possibly because of habitat degradation and environmental pollution, plant diseases and insect pests in the populations were extremely serious, suggesting that the main factors threatening the survival of C. fairylakea populations were not genetic variation, but human activities and the breeding system of this species.     

Genetic assessment of population restorations of the critically endangered Silene hifacensis in the Iberian Peninsula

In order to preserve endangered plant populations and recover their evolutionary potential and ecological behavior, some restoration measures generally involve the reinforcement of the population size in existing natural populations or the reintroduction of new populations. Genetic monitoring of both natural and restored populations can provide an assessment of restoration protocol success in establishing populations that maintain levels of genetic diversity similar to those in natural populations. The highly threatened Spanish species Silene hifacensis (Caryophyllaceae) has only three natural reduced mainland populations in the Iberian Peninsula, following decline and extinction that occurred during the late 20th century. Preterit restoration strategies were essentially based on the implantation of new populations and reinforcement of certain existing populations using transplants mostly cultivated in greenhouses. In the present contribution, levels and patterns of genetic variability within natural and restored populations of Silene hifacensis were assessed using the molecular technique AFLP. Our results pointed out significant genetic diversity differences across the three existing natural populations though their population fragmentation and progressive loss of individuals have not had an impact on the global genetic diversity of this species. For restored populations, their levels of genetic diversity were similar and even higher than in natural populations. As a result, the past restoration protocols were successful in capturing similar and even higher levels of genetic diversity than those observed within natural pools. However, inbreeding processes have been detected for two restored populations. Finally, the main source of plant material for the long-time restored transplants appears to be the natural population of Cova de les Cendres. This study demonstrates, once again, how genetic markers are useful tools to be taken in consideration for endangered plant species conservation plans.     

拟环纹豹蛛不同地理种群遗传多样性的AFLP标记研究

为查明拟环纹豹蛛Pardosa pseudoannulata不同地理种群的遗传多样性机制,应用AFLP技术对6个拟环纹豹蛛地理种群的遗传多样性进行了研究分析。8对引物组合扩增出1 038个AFLP条带,其中多态性条带占86.622%,全部个体显示了各自独特的AFLP图谱。AFLP标记的遗传多样性分析结果表明： 拟环纹豹蛛无论在物种水平(P=86.62%,H=0.2622,I=0.3101),还是在种群水平（P=73.0%,H=0.2155,I=0.2554）都表现出较高的遗传多样性。其中湖南长沙雷锋镇种群内遗传变异最大,云南高黎贡山福贡种群内遗传变异最小,华南北部（湖南、湖北、江西）地区拟环纹豹蛛遗传多样性明显高于华南南部（云南、海南）种群。据种群变异来源分析,有35.77%的遗传变异来自种群间,64.23%的变异来源于种群内（N_m=0.898）,不同地理种群显示出一定的遗传分化。分析认为海拔是影响拟环纹豹蛛遗传分化的重要因素,这为进一步明确我国稻田狼蛛优势种群在农药胁迫下的遗传适应性机制提供了实验依据。     

Conservation genetics of the rare and endangered Leucopogon obtectus (Ericaceae)

Leucopogon obtectus Benth. is a declared rare species found in the kwongan vegetation in Western Australia. Plants on a mineral sand mine and the rehabilitation area are subject to disturbance. Genetic diversity was examined within and among all known populations using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) for conservation. Both molecular markers revealed a high percentage (> 89%) of polymorphic markers and a high mean genetic distance among individuals (D = 0.3). Analysis of molecular variance showed that 86.7% (RAPD) and 89.7% (AFLP) of variability was partitioned among individuals within populations. Exact tests showed no significant population differentiation. The analyses indicated that L. obtectus exhibits high levels of genetic diversity despite small population sizes. The high levels of variability among individuals and the lack of clear population differentiation suggest that this species comprises a single, genetically diverse group. Conservation and management of L. obtectus should concentrate on maintaining the high levels of genetic variability through mixing genotypes and promoting outcrossing.     

Genetic Diversity and Structure of Dalmatian Pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip., Asteraceae) within the Balkan Refugium

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.