Inheritance of random amplified polymorphic DNA (RAPD-PCR) markers and their use in population genetic studies of Calomys musculinus (Rodentia,Muridae), the reservoir of Argentine hemorrhagic fever

In order to assess the reliability of RAPD markers in the estimation of the genetic structure of natural populations of the murid rodent Calomys musculinus (reservoir of Junin virus, ethiological agent of Argentine Hemorrhagic Fever), we have analyzed the heritability of RAPD bands in 10 parents and their offspring (33 individuals). Fourteen out of a total of 119 bands obtained were absent in the parental patterns, but consistently amplified in offspring from some families. These bands can be eliminated from analyses. Overall degree of band sharing between individuals, including non-parental bands, correctly grouped members of a family in the same cluster in a UPGMA tree, with a high bootstrap percentage. Results support the usefulness of RAPDs as hereditable markers. One hundred polymorphic RAPD loci were identified in three natural populations of C. musculinus. Mean expected heterozygosity in three natural populations ranged from 0.206 to 0.220. Allele frequency based and phenotype based measures of genetic differentiation among natural populations of C. musculinus gave similar results (Weir and Cockerham's theta = 0.133; Excoffier et al.'s phi = 0.127). These values were considerably higher than those found previously using allozymes as genetic markers, and are compatible with moderate to low levels of gene flow among populations.     

Haplotype diversity of the mitochondrial DNA D-loop region in Calomys musculinus (Rodentia,Muridae) detected by PCR-RFLP

In order to contribute to knowledge of colonization patterns in the rodent Calomys musculinus, a natural reservoir of the virus producing Argentine hemorrhagic fever (AHF), we studied the haplotype diversity of the mitochondrial DNA D-loop region in five natural populations from central Argentina. Digestion with eight restriction enzymes (RsaI, MseI, Tsp509I, AluI, AciI, HaeIII, NlaIII, and AseI) revealed polymorphism in the 1300 bp fragment amplified by PCR. Twenty different composite haplotypes were detected. Hierarchical analyses indicated that almost all variation (94%) is contained within local populations. Haplotypes 1 and 2, shared by all populations, were the most frequent. Nonsignificant genetic differentiation was found among populations of the endemic and nonendemic areas of AHF: All locations sampled presented exclusive haplotypes in spite of their geographic proximity, which would support previous observations indicating restricted gene flow among C. musculinus populations.     

Genetic differentiation of pedunculate oak Quercus robur L. in the European part of Russia based on RAPD markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73-90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (GST = 0.098); gene flow Nem was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within--population variation.     

大别山山核桃天然群体遗传结构的初步分析

利用RAPD分子标记技术检测了大别山山核桃3个天然居群的遗传多样性和遗传结构。20条10 bp随机引物共检测到238个扩增位点,其中多态性位点162个,多态位点百分率(PPB)为68.1%。居群水平Shannon’s多态性信息指数(I)介于0.2651~0.2801之间;居群水平Ne i’s基因多样性指数(H)介于0.1789~0.1890之间。遗传变异计算显示大别山山核桃居群间基因分化系数(Gst)为0.4063,分子方差分析(AMOVA)表明居群间基因分化水平为0.4177,居群间基因流(Nm)为0.7306,说明大别山山核桃大部分变异存在于居群内,居群间基因交流相对较少。这一结果符合大别山山核桃风媒、异交的繁育系统特点,但其居群间基因分化程度明显高于异交植物的平均水平(Gst=0.1930)。地理隔离、居群内近交及居群间基因流受阻可能是形成目前大别山山核桃天然群体遗传结构的主要因素。     

Recent range expansion and low levels of contemporary gene flow in Calomys musculinus: its relationship with the emergence and spread of Argentine haemorrhagic fever

The geographic distribution of haplotype diversity in the rodent Calomys musculinus, sampled from 16 wild populations of Argentina, was analysed on two geographical scales. The species is the natural reservoir of the Junin virus, the etiological agent of the Argentine haemorrhagic fever (AHF). In all, 24 composite haplotypes were recognised in the mtDNA D-loop region. Haplotypes 1 and 2, internal in the network, were the most frequent and were present in almost all populations. The absence of large genetic gaps between widely distributed haplotypes, the existence of exclusive haplotypes in more than 50% of the sampled populations and the absence of isolation by distance at a macrogeographical scale are in support of the hypothesis of a recent range expansion of the populations of the Humid Pampa, with low to moderate current gene flow. The dispersal of this opportunistic species would have been favoured by the explosive increments in density after agriculture was introduced. When only nearby populations within the endemic area of AHF were considered, a pattern of isolation by distance was detected. At present, genetic drift appears to be the main force acting to randomly differentiate C. musculinus populations, which would also lead to random differentiation of Junin virus strains and a reduction in the virulence of the pathogen in 'historic' AHF areas. The knowledge of migration patterns of the reservoir populations facilitates reliable prediction of the potential spread of the human disease.     

内蒙古高原小叶锦鸡儿(Caragana microphylla)、中间锦鸡儿(C. davazamcii)和柠条锦鸡儿(C. korshinskii)的遗传多样性及遗传关系

采用RAPD技术对10个具较大地理跨度的小叶锦鸡儿、中间锦鸡儿和柠条锦鸡儿种群的遗传多样性和遗传关系进行了研究.共检测到678个位点,多态条带比率(PPB)为100%;特有位点41个,占6.05%.总体上3种锦鸡儿的遗传多样性表现出自东向西递减的趋势,分析表明其与生长地点年均气温呈显著负相关.AMOVA表明:3种锦鸡儿种间变异只占总体变异的6.08%,且显著性检验表明这种变异不显著;种内种群间的变异占总变异的11.90%;总变异的主要部分来自种群内部(82.02%). 3种锦鸡儿各种群总体分析结果表明:种群内变异比率Hpop/Hsp为0.8013,基因分化系数Gst为0.1603,种群每代迁移数Nm为2.6192,显示种群间存在一定强度的基因流,3种锦鸡儿间表现为异交性.3种锦鸡儿多样性高低及种群聚类分布格局都表现出一定的地理连续性.     

Genetic diversity and genetic relationship of Caragana microphylla, Caragana davazamcii and Caragana korshinskii on the Inner Mongolia Plateau, China

C. microphylla, C. davazamcii and C. korshinskii exhibit a geographical replacement series from east to west on the Inner Mongolia Plateau. Currently, there is still a debate about the taxonomic and genetic relationship among these 3 species. We studied the genetic diversity and genetic relationship among these 3 species by analyzing DNA samples of individual plants from within 10 populations with random amplified polymorphic DNA (RAPD) markers. We identified 678 RAPD loci in total, of which all were polymorphic (PPB = 100%). There were 41 unique loci (6.05%). In general, a trend presented that the genetic diversity of these species decreased from east to west. Further, the genetic diversity was significantly negatively correlated with the local annual mean temperature. Analysis of molecular variation (AMOVA) showed that the genetic variation among these 3 species was only 6.08% of the total genetic variation. Between the species, the genetic variation was insignificant (P = 0.9961). The proportion of genetic variation among populations within each species was 11.90% (P < 0.001) of the total genetic variation, and the total genetic variation mainly existed within the populations (82.02%). Estimated with Shannon's index, genetic differentiation within the populations (Hpop/Hsp) was 0.8013, the coefficient of gene differentiation (Gst) was 0.1603, and the gene flow index (Nm) was 2.6192. This, thus, indicates that there is relatively high gene flow among these populations, and that these 3 species are crossbreeding. The genetic diversity level and the population distribution pattern showed geographic continuity to some extent.     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

有性生殖对栗疫病菌群体结构的影响

采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD随机引物中筛选出扩增多态性丰富的4条引物,共扩增出条带73条,多态性检测率为100%。研究结果表明,在8个子囊壳和无性生殖群体中的基因多样性,64.27%由群体内部引起,只有35.73%的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389,基因流动值为3.4212,说明子囊壳群体和无性生殖群体之间存在一定的系统关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界的病菌的多样性起重要的作用。     

Genetic variability and differentiation of Caragana microphylla populations as revealed by RAPD markers

Genetic variability in random amplified polymorphic DNA (RAPD) was studied in 90 individuals of Caragana microphylla, an outcrossing perennial shrub species, from five natural populations sampled in Inner Mongolia steppe of China on a small scale. Nineteen selected primers were used to amplify DNA samples, and totally 225 bands were detected. The percentage of polymorphic bands within populations ranged form 58.22% to 63.56%, with an average of 60% at the population level and 71.11% at the species level, indicating relatively high genetic variations in C. microphylla species. Shannon's information index (I) and Nei's gene diversity (h) showed the similar trend with each other. According to the analysis of Nei's gene diversity, the percentage of genetic variation among populations was 7.13%, indicating a low level of genetic differentiation among populations. There existed a strong gene flow (Nm = 3.26) among populations. Although AMOVA analysis also revealed most variation was within populations (phi(ST) = 4.1%), a significant proportion was observed among populations (P<0.001) in the present study, suggesting genetic differentiation occurred among populations at a certain extent. Based on Mantel's tests and the results of previous studies, the genetic structure pattern of C. microphylla accorded with the isolation-by-distance model on a very large scale, however, on a small scale, the significant genetic differentiation among populations might be enhanced by the micro-environmental divergence among the sampling sites, rather than by geographic factors. Analysis of the genetic variations of C. microphylla populations provided useful information for the adaptive strategy of Caragana species.     

亚欧美栗疫病菌群体的遗传多样性

从 12 0个随机引物中筛选出条带清晰、主带明显、重复性好的 9个引物 ,对来自不同地域和寄主的 7个群体的 14 2个栗疫病菌菌株进行 RAPD分析。 9个引物共扩增出条带 12 4条 ,其中多态性条带 111条 ,多态性比率为 89.5 2 %。利用 Popgen3.2软件对供试群体进行遗传多样性分析和 UPGMA聚类。结果表明 ,中国地区 4个群体间的遗传相似性较大 ,与美国、意大利和日本群体间的相似性较小 ;美国和意大利群体间的遗传相似性较大 ,且它们与日本群体间的相似性大于与中国群体间的相似性。病原菌群体的遗传变异率为 0 .2 35 1,其中在地区水平上 ,82 .34%由群体内的变异引起 ,17.6 6 %由群体间的差异引起 ,群体间的基因流动值为 2 .3311;而在寄主水平上 ,则 79.4 2 %由群体内的变异引起 ,2 0 .5 8%由群体间的差异引起 ,群体间的基因流动值为 1.92 97     

苹果属山荆子遗传多样性的RAPD分析

采用RAPD分子标记对东北、华北地区山荆子8个天然居群的137株个体进行了遗传多样性研究,10个引物共得到72个扩增位点,其中多态性位点63个.多态位点百分率为87.50%,有效等位基因数(Ne)为1.602 1,Nei's基因多样性(H)为0.338 6,Shannon信息指数(J)为0.496 1,表明山荆子遗传多样性水平较高.基因流(Nm)为1.735 3,说明山荆子各居群间存在一定的基因交流.居群间基因分化系数(Gst)值为0.223 7,说明虽然山荆子居群的遗传变异主要存在于居群内,但各居群间也存在着较高的遗传分化.     

Genetic Differentiation of Pedunculate Oak Quercus robur L. in the European Part of Russia Based on RAPD Markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73–90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (G _ST = 0.098); gene flow N _e m was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within-population variation.     

濒危灌木长叶红砂遗传多样性的RAPD分析

应用RAPD分子标记对濒危灌木长叶红砂(Reaumuria trigyna)种群遗传多样性进行了分析.应用18条随机引物对长叶红砂5个种群的95个个体进行扩增,检测到118个位点,其中多态位点105个.结果表明:长叶红砂种群的多态位点比率(P)为88.98%,显示出长叶红砂种群存在较高的遗传多样性.Shannon多样性指数(0.4966)、Nei基因多样性指数(0.3303)和基因分化系数(Gst=0.1425)的分析结果显示,长叶红砂种群遗传变异大多存在于种群内,种群间的遗传分化占14.25%.聚类分析表明,长叶红砂种群遗传距离与地理距离之间无直接相关关系.遗传多样性水平与物种特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低.     

地被植物鹅绒委陵菜的遗传多样性研究

采用RAPD方法对不同海拔和植被盖度下的鹅绒委陵菜（Potentilla anserinaL.）6个种群的遗传多样性水平进行了研究。结果表明,13条随机引物共扩增出132条带,多态性位点为117;鹅绒委陵菜物种水平的遗传多样性较高,且种群间分化明,多态位点比率（88.64%）、Nei’s基因多样性（0.3180）、Shannon信息多样性指数（0.4732）均远高于多数克隆植物。6个种群内的遗传多样性水平变化较大,多态位点在各种群中分布不均衡,种群间分化系数（GST）为47.7%,基因流较低（Nm=0.5482）,多态位点比率在31.06%～74.24%之间,Shannon信息指数在0.1711～0.3625之间,Nei基因多样性指数在0.1164～0.2425之间。多样性水平的变化与海拔没有明显的相关性,而与生境盖度呈显著正相关。从而推断在盖度低、资源丰富的环境中,该物种可能更倾向于克隆繁殖。     

内蒙古典型草原羊草种群遗传分化的RAPD分析

运用 RAPD技术对内蒙古典型草原不同生境 8个羊草种群进行分析。采用 2 4个随机引物 (10 nt)在 8个种群中共检测到2 2 4个扩增片断 ,其中多态性片断 173个 ,总的多态位点百分率达 77.2 % ,特异性片断 2 2个 ,占 9.82 % ,平均每个引物扩增的DNA带数为 9.3 3条。利用 Nei指数和 Shannon指数估算了 8个种群的遗传多样性 ,并计算种群相似系数和遗传距离 ,运用UPGMA法进行聚类分析。结果表明 :羊草大部分的遗传变异存在于种群内 ,只有少部分的遗传变异存在于种群间 ,Nei指数和Shannon指数计算结果分别为 85.4%和 66.8% ;羊草不同种群的遗传多样性存在差异 ;8个羊草种群平均遗传距离为 0 .2 3 16,变异范围为 0 .1587～ 0 .2 70 0 ,说明 8个羊草种群间的遗传变异不大 ,即 :在较小地理范围内羊草的遗传分化程度较小 ;8个种群可聚为 3个类群 ,聚类结果显示生境相似的种群能够聚在一起 ,而地理距离最近的种群不一定归为一类 ,说明小范围内羊草种群间的遗传分化与地理距离不存在相关性 ,而与其生境间的相似度相关。影响遗传相似性的不是单一因子而是各种因子的综合作用 ,较小地理范围内羊草种群间的遗传分化主要是由环境的异质性所引起的     

长筒石蒜居群遗传多样性RAPD分析

利用RAPD标记对长筒石蒜3个居群的遗传多样性及分化程度进行了研究。12条随机引物扩增出94个可分析位点,多态位点比率(PPB)为65.96%,表明长筒石蒜具有比较高的遗传多样性。经POP-GENE32分析表明:Nei’s基因多样性指数(h)为0.1897,香农多样性指数(Ⅰ)为0.2945,基因分化系数(GST)为0.1191,基因流(Nm)为3.6980。经WINAMOVA分析表明:居群内遗传变异占71.75%,而居群间只占28.25%。遗传多样性分析表明,各居群的遗传多样性水平由高到低为琅琊山居群>宝华山居群>盱眙居群。遗传分化表明:长筒石蒜各居群间遗传分化程度较低;大部分遗传变异存在于居群内部,表明其具有较强的进化潜力,自然情况下不会处于濒危状态,野生种质资源的破坏,主要来自于人为干扰。     

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 Φ _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 θ_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.     

RAPD analysis of genetic diversity and population genetic structure of Stipa krylovii Reshov. in Inner Mongolia steppe

Random amplified polymorphic DNA (RAPD) analysis was used to characterize the genetic diversity and population genetic structure of Stipa krylovii populations in Inner Mongolia steppe of North China. Thirteen 10 bp oligonucleotide primers, which generated 237 RAPD bands, were used to analyze 90 plants of five populations from three regions, meadow steppe, typical steppe and desert steppe, from the east to the west. The genetic diversity of Stipa krylovii that was revealed by observed number of alleles (na), expected number of alleles (ne), Nei's diversity index (h), Shannon's diversity index (H), amplificated loci, polymorphic loci and the percentage of polymorphic loci (PPB) increased from the east to the west. The Pearson's correlation analysis between genetic diversity parameters and ecological parameters indicated that the genetic diversity of Stipa krylovii was associated with precipitation and cumulative temperature variations along the longitude (humidity were calculated by precipitation and cumulative temperature). Dendrogram based on Jaccard's genetic distance showed that the individuals from the same population formed a single sub-group. Although most variation (56.85%) was within populations, there was high genetic differentiation among populations of Stipa krylovii, high differentiation within and between regions by AMOVA analysis. Either Nei's unbiased genetic distance (G(ST)) or gene flow (Nm) among pairwise populations was not correlated with geographical distance by Mantel's test (P > 0.05), suggesting that there was no consistency with the isolation by distance model in these populations. Natural selection may have played a role in affecting the genetic diversity and population structure, but habitat destruction and degradation in northern grassland in China may be the main factor responsible for high genetic differentiation among populations, within and among regions.     

Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis

Isoetes coreana Chung and Choi is on the list of critically endangered species in South Korea. Using randomly amplified polymorphic DNA (RAPD) markers, we investigated the genetic diversity within and between seven local populations of I. coreana. Ten RAPD primers produced a total of 94 bands, of which 59 (62.8%) were polymorphic. A low level of genetic diversity was recognized within the populations of I. coreana: polymorphic loci (P), with values ranging from 3.4% to 33.9%, and a mean value of 15.5% being observed. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater among populations (81.6%) than within populations (19.7%) or among the three regions included in this study (Han River, Youngsan River, and Nakdong River). In addition, a high degree of genetic differentiation (θ^B = 0.742) was detected among the populations. These results indicate that genetic differentiation has occurred very rapidly. However, the rate of gene flow between populations was found to be as low as 0.059, irrespective of the genetic and geographical distances between the populations, which indicates that genetic drift must have played an important role in forming the present populations of I. coreana. Because a reduction of genetic diversity as a result of genetic drift is undesirable, increasing the gene flow between populations of Korean quillwort I. coreana should be considered as a conservation strategy.