我国西南部喀斯特森林特有濒危树种掌叶木新的微卫星分子标记的开发

采用基因组DNA富集文库法FIASCO(Fast isolation by AFLP of sequences containing repeats)从掌叶木(Handeliodendron bodinieri)基因组中分离和筛选了10个新的微卫星位点,进而对掌叶木茂兰自然分布居群的遗传多样性进行了分析。结果表明, 每个位点在30株掌叶木个体上的平均等位基因数(A)为3.5(3~5个),平均观察杂合度(H_O)为0.650(0.267~0.900),平均预期杂合度(H_E)为0.494(0.224~0.652)。每个位点的第一排除概率值P_r(Ex₁)为0.029~0.240,位点综合值为0.7496。单个位点的第二排除概率P_r(Ex₂)为0.123~0.419,位点综合值为0.9517。这些信息预示着这些微卫星标记可以为研究喀斯特特有濒危树种掌叶木的基因流及居群遗传结构提供有效的遗传工具。     

濒危特有种掌叶木的微卫星遗传多样性研究

掌叶木居群具有较丰富的遗传多样性,该研究利用9对微卫星(SSR)分子标记揭示了掌叶木(Handeliodendron bodinieri)的遗传多样性。结果表明:观测等位基因数(Na)平均为3.903,有效等位基因数(Ne)平均为2.545,期望杂合度(He)平均为0.521,Shannon’s多态性信息指数(I)为0.962,PIC平均值为0.465。掌叶木的自然分布居群有相对较高的遗传多样性,但由于人为破坏等因素导致该群体濒危,而濒危并不是因为遗传多样性降低而造成的。居群间的遗传分化为掌叶木8个居群间的遗传一致度为(GI=0.849~0.970),遗传距离为(GD=0.032~0.164)。基于Nei’s遗传距离用UPGMA法对掌叶木居群进行聚类,Nei’s的基因分化系数为(G_(st))为0.027,平均Nei标准遗传分化系数(G'_(st)N)为0.031,平均Herick’s标准遗传分化系数(G'_(st)H)为0.064,基因流(N_m)为3.368。AMOVA分析结果表明:掌叶木居群间变异占3%,居群内变异占97%,居群内的遗传分化大于居群间的分化。利用Mantel检测发现,居群间的遗传距离与地理距离显著正相关(r=0.299,P0.05)。该研究结果为掌叶木生物多样性和资源保护与利用提供了更充分的科学依据。     

Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in Hungary

In this study, we used genetic-based approaches to estimate population size and structure of Eurasian otter along the Drava River in Hungary, and compared these results to traditional survey-based methods. The relative spraint density of otter was estimated based on the number of fresh (D_f) and total number (D_t) of spraints collected on standard routes over a 2-year period. Nine microsatellite loci were screened, generating 17 individual otter genotypes composed of 45 different alleles. The expected heterozygosity ranged from 0.53 to 0.89 and observed heterozygosity from 0.25 to 0.92. The mean density (D_g) estimated over six different sites was 0.17 individuals per km of shoreline. A close correlation was found between the number of genotypes and spraint counts along a standard route (fresh spraints: r_P=0.85, P<0.01; total spraints r_P=0.76, P<0.05). All genotypes found within the 50 km-long study area were closely related (D_m ranged between 0.08 and 0.21).     

Eight SSR loci from Oyster Crassostrea gigas EST database and cross-species amplification in C. plicatula

Oyster (Crassostrea plicatula) is widely distributed in coastal areas of China. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Crassostrea gigas and to amplify EST-SSR in C. plicatula. Characteristics of eight EST-SSR loci were investigated using 37 wild-type C. plicatula individuals. The number of alleles per locus ranged from four to six. The observed heterozygosity (H _O) ranged from 0.1892 to 0.7027 (0.3919 on average) and the expected heterozygosity (H _E) ranged from 0.6068 to 0.7656 (0.7039 on average) (P < 0.05). The average observed heterozygosity was much lower than the average expected heterozygosity. All loci except C15 departed from Hardy-Weinberg equilibrium (P < 0.05) significantly. Contribution of the eight EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the species of oysters in the future.     

蚯蚓对废纸屑再利用及养分贫瘠土壤综合质量的影响

办公废纸屑作为常见有机废弃物,由于体积小且转化为再生纸成本高,因而再利用很难。但其含有大量有机碳(特别是纤维素)可能有助于退化土壤修复。蚯蚓对土壤有机质分解和其他土壤功能有重要影响,办公废纸屑和蚯蚓共同作用如何影响养分贫瘠土壤质量至今未知。研究以赤子爱胜蚓为接种蚯蚓,将办公废纸屑添加到养分贫瘠土壤中,分别设置纯土壤培养为对照组(S)、单独添加废纸屑(SP)、单独接种赤子爱胜蚓(SE)和添加废纸屑并接种赤子爱胜蚓的处理(SPE),比较培养90 d后各处理理化指标(pH、有机碳、全氮、全磷、全钾、碱解氮、速效磷、速效钾、交换性阳离子钾、钠、钙、镁等)、微生物磷脂脂肪酸(PLFAs)总含量和微生物结构的差异,在此基础上综合评价土壤质量,阐明废纸屑和赤子爱胜蚓在养分贫瘠土壤改良修复中的作用。结果显示：SPE处理较SP处理显著提高废纸屑的分解率89.48%。与对照相比,SP处理能够显著提高土壤pH值2.94个单位,SPE处理能够使其维持在中性水平;前者显著提高土壤有机碳(SOC)125.76%,交换性钠钙镁(Na_Ex、Ca_Ex、Mg_Ex     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

Isolation and characterization of microsatellite loci in Eurycorymbus cavaleriei,a dioecious endemic tree species in China

Eleven dinucleotide microsatellites were developed and characterized for Eurycorymbus cavaleriei, a dioecious, rare and endemic tree species in China. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 30 trees from two natural populations. The observed heterozygosity (H_O) ranged from 0.379 to 0.931, with an average of 0.625. These markers provide powerful tools for the ongoing population and conservation genetics studies of E. cavaleriei.     

Isolation of highly polymorphic autosomal microsatellite loci and a sex‐linked locus from sugarbirds

We describe the isolation of six tetranucleotide microsatellites from the Cape sugarbird (Promerops cafer) using an enrichment protocol. All loci were highly variable with number of alleles ranging from nine to 26 and values of observed heterozygosity ranging from 0.534 to 0.931. All loci were in Hardy–Weinberg equilibrium with the exception of Pro66 and Pro86. Further analysis of Pro86 indicated it was Z‐linked. All loci amplified and were variable in the congeneric Gurneys sugarbird (Promerops gurneyi). These loci will be used in mating system and phylogeographic studies of Cape sugarbirds in South Africa.     

土壤石砾含量对掌叶木幼苗生长和根系特征的影响

为探讨土壤石砾含量对珍稀濒危植物掌叶木幼苗生长和根系的影响,该研究以1个月生掌叶木幼苗为试验材料,进行5种不同土壤石砾含量[0(CK),20%,40%,60%和80%]盆栽试验,筛选最适宜掌叶木幼苗生长的土壤石砾含量。结果表明:(1)土壤石砾含量对掌叶木幼苗生长有极显著影响,其中幼苗苗高和地径相对增长率、叶面积、苗木质量指数、生物量(根、叶和全株)和根冠比均在土壤石砾含量为40%时最大。(2)土壤石砾含量对掌叶木幼苗根系形态具有极显著影响,总根长和根表面积在土壤石砾含量为40%时最大; 而根系平均直径随石砾含量增加逐渐减小,当土壤石砾含量高达80%时,根系平均直径最小。(3)土壤石砾含量对掌叶木幼苗根系拓扑结构和分形特征无显著影响,而对根系平均连接长度和分叉数有极显著影响,其中各处理根系拓扑指数(TI)、修正拓扑指数(q_a和q_b)均趋近于1,即掌叶木幼苗根系在不同土壤石砾含量中分支模式更趋近于鱼尾形分支; 根系平均连接长度随石砾含量增加先增大后减小,在土壤石砾含量40%时最大; 根系分叉数随石砾含量增加逐渐减小。(4)综合评价幼苗生长和根系形态与构型指标表明,掌叶木幼苗在40%土壤石砾含量中地下根系和地上茎叶生长状况最好。因此,土壤添加适量石砾能促进掌叶木幼苗生长,当石砾含量为40%时幼苗生长效果最好,苗木质量指数最高,最适宜掌叶木幼苗生长。     

Population genetic structure of <Emphasis Type="Italic">Sargassum thunbergii</Emphasis> (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 F _ST 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.     

猴樟叶绿体基因组特征分析

猴樟(Cinnamomum bodinieri)枝叶含有丰富的精油,是重要的园林绿化树种和经济树种,但目前有关猴樟基因组学的研究报道不多。为揭示猴樟叶绿体基因组特征及系统发育关系,该文基于高通量测序平台进行测序,从头组装了完整的猴樟叶绿体基因组,并对其基因组结构、基因构成及序列重复、密码子使用偏好性以及系统发育进行分析,结合樟亚科主要属物种叶绿体基因组数据构建系统发育树。结果表明:(1)猴樟叶绿体基因组全长152 727 bp,包括一对20 132 bp的反向重复(IRs)区、93 605 bp的大单拷贝(LSC)区和18 858 bp的小单拷贝(SSC)区,总GC含量为39.13%。(2)该基因组共编码127个基因,包括83个蛋白质编码基因(PCGs)、36个转运RNA基因(tRNAs)和8个核糖体RNA基因(rRNAs); 共鉴定出92个SSR位点,其中大部分是A/T组成的单核苷酸重复序列; 密码子适应指数(CAI)为0.166,有效密码子数(ENc)为54.68; 猴樟与近缘种的叶绿体基因组主要在IR区和2个SC区边界上存在一定的差异。(3)24种樟亚科植物的系统发育树显示,猴樟与樟树亲缘关系最近,同时支持了樟属-甜樟属分支(Cinnamomum-Ocotea Clade)、月桂属-新木姜子属分支(Laurus-Neolitsea Clade)、润楠属-鳄梨属分支(Machilus-Persea Clade)的建立。该研究丰富了猴樟遗传资源信息,进一步确定了樟亚科主要属的系统发育地位。     

Genetic Structure of Blastocerus dichotomus Populations in the Paraná River Basin (Brazil) Based on Protein Variability

The population structure of 147 marsh deer (Blastocerus dichotomus) from three areas in the Paraná River basin, Brazil, was studied by observing protein polymorphism at 17 loci. Six loci were polymorphic and 11 monomorphic. The proportion of polymorphic loci (P) was 35.29% and the average heterozygosity (H) was 6.31%. Wright's F_ST indicated that only 4.9% of the total variation in allelic frequencies was due to genetic differences between the three groups. The high value of F_IS (0.246) indicated inbreeding in the marsh deer. Genetic distance values (D = 0.014–0.051) showed little divergence between the three areas. We suggest that probable mechanisms accounting for the genetic structure are female phylopatry and polygyny and also that inbreeding has resulted from decreasing areas of wetland leading to isolation, overhunting, and diseases transmitted by cattle.     

Isolation and characterization of microsatellite loci in Pratt’s Leaf-nosed Bat (<Emphasis Type="Italic">Hipposideros pratti</Emphasis>) and cross-species amplification in closely related taxa

We developed nine microsatellite loci using an enriched library method from the genomic DNA of Pratt’s leaf-nosed bat (Hipposideros pratti). These loci were tested on 96 individuals sampled from Sichuan Province, China. The number of alleles per locus varied from 4 to 14. The expected and observed heterozygosity values ranged from 0.513 to 0.886 and from 0.375 to 0.966, respectively. Three microsatellite loci departed significantly from Hardy–Weinberg equilibrium (HWE). No linkage disequilibrium was found. These microsatellite loci will be used in studies of conservation genetics in this species.     

Isolation and characterization of highly polymorphic microsatellite loci in Schreibers’ long‐fingered bat,Miniopterus schreibersii (Chiroptera: Vespertilionidae)

Five polymorphic, dinucleotide microsatellite loci have been identified and characterized in Schreibers’ long‐fingered bat, Miniopterus schreibersii (Chiroptera: Vespertilionidae). These include three uninterrupted (CA)_n repeats, ranging in length from (CA)₁₇ to (CA)₁₉, one interrupted repeat (CA)₄(CG)₂(CA)₁₃, and one uninterrupted (GA)₂₇ repeat. All loci were highly polymorphic, with 17–20 alleles identified per locus. Observed heterozygosity levels (0.66–0.82) are lower than expected due to homozygote excess, probably caused by pooling of populations, resulting in population substructuring. All five polymorphic loci were also successfully amplified in the closely related M. fraterculus. Two pairs of primers additionally amplified polymorphic microsatellites in Chaerophon sp.     

<Emphasis Type="Italic">Tiliqua rugosa</Emphasis> microsatellites: isolation via enrichment and characterisation of loci for multiplex PCR in <Emphasis Type="Italic">T. rugosa</Emphasis> and the endangered <Emphasis Type="Italic">T. adelaidensis</Emphasis>

We used an enrichment technique to isolate 18 novel di and tri microsatellites for the socially monogamous lizard Tiliqua rugosa. These loci were amplified in conjunction with previously described loci in two and three PCR multiplexes for T. rugosa and the endangered T. adelaidensis, respectively. The loci were highly polymorphic in both species, exhibiting between 2 and 32 alleles with observed heterozygosity ranging from 0.43 to 0.96. These markers will be useful for population-level analyses and can contribute to a genetic foundation for conservation strategies for the endangered T. adelaidensis.     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

Allozyme Variation and Phylogenetic Relationships in Picea jezoensis (Pinaceae) Populations of the Russian Far East

Genetic variation and differentiation of 12 populations of Picea jezoensis from the Russian Far East were studied using 20 allozyme loci. The mean number of alleles per locus was 2.63, the percent of polymorphic loci was 88.1%, the observed heterozygosity was 0.181, and the mean value of expected heterozygosity amounted to 0.189. The values of expected heterozygosity of the northern and central mainland populations were higher than in the southern part of the natural range. A significant bias of Hardy–Weinberg heterozygosity to equilibrium heterozygosity (H_eq) suggests that most of the mainland populations have recently experienced a severe expansion in population size while populations from Kamchatka Peninsula have undergone a reduction in population size. Unbiased Nei’s genetic distance values were low within and between the mainland and Sakhalin Island populations (D_N=0.008). The largest values (D_N=0.063) were found between the mainland/Sakhalin and Kamchatka Peninsula populations. Based on genetic distance, P. jezoensis and P. kamtschatkensis could be considered as distinct taxa, but P. ajanensis, P. microsperma, and P. komarovii do not warrant taxonomic recognition.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.