三叶木通微卫星分子标记开发及评价

为了获得适于三叶木通遗传多样性和遗传结构研究的微卫星分子标记,该研究采用磁珠富集法构建了三叶木通微卫星富集文库。结果表明:在150个阳性克隆中发现了70个微卫星位点,富集效率为46.67%,其中含双碱基重复单元的序列占比为79.37%,三碱基和四碱基重复含有量较少。共设计引物63对,其中筛选出16对高多态引物,对1个三叶木通自然居群48个个体进行了遗传分析,结果显示位点的等位基因数为10~22个,观察杂合度和期望杂合度分别为0.370~0.792和0.724~0.936,多态性信息指数为0.725~0.919,表明以上引物均为高多态性引物。其中,12个位点偏离哈迪-温伯格平衡,呈现出纯合子过剩状态,这可能与哑等位基因和其它因素有关。综上结果表明,该研究所开发的16对引物能够用于三叶木通遗传多样性和遗传结构评价工作。     

Assessment of genetic variability among selected species of Apocynaceae

Family Apocynaceae is an economically important family grown as ornamental plants and many wild species have medicinal uses as well. The aim of the present study was to understand the level and pattern of genetic variability among the selected individuals of Apocynaceae. For this purpose, three species of different genera of Apocynaceae, Thevetia peruviana, Alstonia scholaris and Catharanthus roseus, were collected from Rawalpindi and Quaid-i-Azam University forest, Islamabad. To evaluate the level of polymorphism within the species and members of different species, randomly amplified polymorphic DNA (RAPD) markers were used. A series of OPC RAPD primers were used; only six primers of OPC series gave amplification. Highest genetic variation at interspecific and intraspecific levels was shown by OPC 9 and the lowest polymorphism was observed in OPC 4. The data was analyzed by using software Statistica 5.5. In total 105 monomorphic and 272 polymorphic bands were produced from all primers. Therefore, out of 322 amplified products, 26% were monomorphic and 68% were polymorphic. Low genetic diversification was observed both at intraspecific and interspecific level. At the molecular level Alstonia scholaris and Catharanthus roseus (subfamily Plumerioideae) appeared in a group and Thevetia peruviana (subfamily Rauvolfoideae) formed another group, confirming the classification based on morphological characters.     

中国重要农业害虫韭菜迟眼蕈蚊多态性EST-SSR标记的开发

【背景】韭菜迟眼蕈蚊是我国重要的农业害虫,然而它的遗传资源有限。本研究旨在开发韭菜迟眼蕈蚊EST-SSR标记,为研究不同地区的韭菜迟眼蕈蚊种群结构和遗传多样性奠定基础。【方法】从韭菜迟眼蕈蚊的表达序列标签(EST序列)中设计16对简单重复序列(SSR)引物,进一步筛选出9对具有多态性的SSR引物。【结果】从42095条unigene中确定了3383个SSR位点。利用查找到的SSR位点共设计出16对引物,进一步检测筛选发现9对引物具有多态性,引物的每个位点平均有3.33个等位基因。利用9对引物对30头韭菜迟眼蕈蚊进行检测,共获得30个等位基因,观测杂合度和期望杂合度的范围分别为0.0000~0.6875和0.0370~0.6877;其中,9个位点中有5个位点显著偏离Hardy-Weinberg平衡。【结论与意义】本研究成功从迟眼蕈蚊EST序列中筛选出9个具有多态性的微卫星位点,这为进一步分析该害虫种群的遗传结构和遗传多样性奠定了基础。     

Thirteen polymorphic microsatellite DNA loci from whiptails of the genus Aspidoscelis (Teiidae: Squamata) and related cnemidophorine lizards

We describe polymerase chain reaction primers and amplification conditions for 13 microsatellite DNA loci isolated from two bisexual species of whiptail lizards Aspidoscelis costata huico and Aspidoscelis inornata. Primers were tested on either 16 or 48 individuals of A. c. huico and/or 26 individuals of A. inornata. Ten of the 13 primers were also tested against a panel of 31 additional whiptail taxa. We detected three to nine alleles per locus in A. c. huico and four to 19 alleles per locus in A. inornata, with observed heterozygosity ranging from 0.60 to 0.87 and from 0.15 to 1.00, respectively. These primers will be an important resource for surveys of genetic variation in these lizards.     

Molecular Characterization of a Variable Tandem Repeat Sequence Determined during RAPD Analysis among Posidonia oceanica Insular and Coastline Populations

The seagrass Posidonia oceanica plays a multifunctional role in the coastal area as an important and productive component of ecosystems in the Mediterranean Sea. We detected by RAPD analysis with two arbitrary primers genetic differences in P. oceanica collected from several sites in the Southern Mediterranean. By AMOVA analysis we observed a level of about 20% genetic difference among individuals within a population and 80% among populations. A common band of 200 bp was found in all the amplified samples. Cloning and sequencing analysis of this band revealed the presence of a simple tandem repeat sequence (minisatellite) that we called PoTR (Posidonia oceanica tandem repeat). Finally, the ability of PoTR to detect genetic variability inP. oceanica genome was demonstrated by the presence of amplification products of different lengths utilizing primers internal to this sequence.     

Polymorphic microsatellites in the African freshwater snail,Bulinus forskalii (Gastropoda,Pulmonata)

Eleven microsatellites were isolated in the freshwater snail Bulinus forskalii, intermediate host for the medically important trematode Schistosoma intercalatum. Characterization in 60 snails from three populations of B. forskalii from Cameroon revealed 4 to 18 alleles per locus. Low observed heterozygosity but higher expected heterozygosity, high F_IS estimates, significant departures from Hardy–Weinberg equilibrium and genotypic linkage disequilibria all indicate that B. forskalii is a preferential selfer. High F_ST estimates suggest that effective dispersal is limited and genetic drift is an important determinant of genetic structure. The potential utility of the microsatellite primers in other closely related Bulinus species was explored.     

Microsatellite loci from the Caribbean Fruit Fly,Anastrepha suspensa (Diptera: Tephritidae)

The lack of polymorphic genetic markers suitable for genotyping sperm, eggs, and all life stages of the important agricultural pest, Anastrepha suspensa, have prevented detailed genetic studies of its breeding system, reproductive dynamics, and population dynamics. We describe polymerase chain reaction (PCR) primers and reaction conditions for amplifying nine polymorphic microsatellite DNA loci isolated from this species. The PCR primers were tested on four to five individuals each collected from five geographically distant locations in Florida. Heterozygosity values and the number of alleles per locus varied from 0.11 to 0.89, and from two to 12, respectively.     

Genetic diversity and population structure of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bge in China revealed by ISSR and SRAP

Salvia miltiorrhiza Bge is a traditional Chinese medicinal herb used as an important drug to cure cardiovascular diseases. In this work, inter simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP) markers, were applied to assess the level and pattern of genetic diversity in five important cultivated populations of S. miltiorrhiza. Among these populations, 120 bands were amplified by 5 ISSR primers, of which all were polymorphic, and 110 polymorphic bands (90.16%) were observed in 122 bands amplified by 6 SRAP primers. A high levels of genetic diversity at the species level was detected with Hs = 0.1951, 0.1927 respectively. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (86.64 and 84.83% respectively) rather than among populations (13.36 and 15.17% respectively). Cluster analysis divided the five populations into two groups. The genetic relationships among populations have low correlation with their geographical distribution (Mantel test; r = 0.4870 and 0.5740 respectively). The study indicated that both ISSR and SRAP markers were effective and reliable for assessing the degree of genetic variation of S. miltiorrhiza. Our results suggested that random collecting, preserving and planting seeds without deliberate selection might be an efficient way to conserve genetic resources of medicinal plants. Their effective use was also discussed on the further breeding.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Isolation and characterization of microsatellite loci from mangrove red snapper Lutjanus argentimaculatus

Lutjanus argentimaculatus, also called mangrove red snapper, is a commercially important fish in East Asia. A proper understanding of population structure is primarily linked with the management of genetic resources in exploiting marine fisheries. Herein, seven microsatellite loci, which showed high polymorphism (observed heterozygosity per locus ranging from 0.3571 to 0.7857 and expected heterozygosity per locus ranging from 0.6236 to 0.8821), were isolated and characterized from L. argentimaculatus. Cross‐species amplifications also indicate that primers designed for these loci may be useful for further studies about other closely phylogenetic species of the family Lutjanidae.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.     

Genetic diversity in inter‐simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.), in the monogeneric family Punicaceae, is found in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary centre of origin. In India, pomegranate occurs naturally only in the Western Himalayan regions of Jammu and Kashmir, Himachal Pradesh and Uttarakhand States. However, there is no information about genetic variation in wild pomegranate at population level. In this paper, we describe genetic diversity across natural populations of Indian pomegranate based on inter‐simple sequence repeat (ISSR) markers. Forty‐nine accessions representing eight populations from two regions were analysed using ISSR. Seventeen ISSR primers resulted in 268 polymorphic bands, with 87.01% polymorphism throughout the accessions. Pair‐wise population genetic distances ranged from 0.05 to 0.45, with a mean of 0.25 between populations. amova and Nei’s genetic diversity analyses revealed higher genetic variation within populations than among populations. A higher genetic differentiation (G_ST) was observed between the spatially distant populations, indicating a low level of genetic exchange (Nm) among these populations. However, clustering of populations was not in accordance with their geographical affiliations in the tree. The results indicate that the ISSR method is sufficiently informative and powerful to assess genetic variability in pomegranate, and that patterns of genetic variability observed among populations of wild pomegranate from the Western Himalaya differ. Estimation of genetic variation reported here provides a significant insight for in situ conservation and exploitation of genetic resources for this economically important species as potential breeding material.     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

Isolation of microsatellite loci in the codling moth,Cydia pomonella (Lepidoptera: Tortricidae)

Cydia pomonella L. is an important insect pest of pome fruits worldwide. We have isolated and characterized 17 microsatellite loci from the enriched genomic libraries constructed using a biotin/streptavidin capture protocol. Among these loci, 11 were scored polymorphic when 50 individuals from a laboratory population were screened. Their alleles numbered two to four, with the observed heterozygosity (H_O) ranging from 0.114 to 0.404. Successful amplification was obtained for all these loci when the designed primers were tested, showing the promise of use in genetic mapping and population studies.     

Development and characterization of microsatellite markers for Chinese bayberry (Myrica rubra Sieb. & Zucc.)

Chinese bayberry (Myrica rubra Sieb. & Zucc.) is one of the most important fruit tree crops in southern China. This paper reports the development of microsatellite primers for Chinese bayberry. By screening the expressed sequence tag database and other sources, 11 polymorphic loci were generated and primers were designed. Polymorphism of these 11 loci was assessed in 32 cultivars. All the 11 loci are polymorphic and the number of alleles (A) ranged from 3 to 12, observed heterozygosity (H _O) and expected heterozygosity (H _E) ranged from 0.1250 to 0.9667 and 0.2359 to 0.8790, respectively, polymorphism information content ranged from 0.2285 to 0.8516. These microsatellite loci should be useful in the studies of genetic diversity of Myrica rubra and will provide useful implications for resource conservation.     

基于SSR分子标记的中国黄连木遗传多样性分析北大核心CSCD

该研究利用筛选出的7对SSR引物,对中国20个省、市、自治区的210份种质资源进行分子标记试验,分析中国黄连木种质资源遗传多样性、亲缘关系、遗传分化特点并构建DNA分子身份证,为黄连木的资源保护、种质利用提供理论依据,结果表明:(1)7对引物在210份种质中共扩增出158个等位基因位点,平均每对引物的等位基因数为22.571个。(2)基因多样性(GD)变化幅度为0.654~0.913,平均为0.804;期望杂合度(He)变化范围0.257~0.771,平均为0.532;多态信息含量(PIC)变化范围0.639~0.907,平均为0.784。(3)从不同地区黄连木群体的遗传多样性来看,观测杂合度(Ho)介于0.373~0.600之间,平均值为0.520;期望杂合度(He)介于0.632~0.811之间,平均值为0.737;从各群体间遗传分化指数(Fst)来看,黄连木各地区群体间的遗传分化值在0.015~0.099之间,各群体间的遗传分化处于中等以下水平。(4)分子方差分析(AMOVA)结果显示,黄连木的遗传分化变异以群体内为主,占总变异量的94%,群体间的变异占6%。(5)UPGMA聚类、群体遗传结构分析和PCoA分析结果相一致,全部种质被划分为两大类,西南地区群体单独为一类,其他地区单独为一类。(6)利用7对SSR引物构建了210份黄连木种质的DNA分子身份证。     

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.     

Evaluation of genus <Emphasis Type="Italic">Cenchrus</Emphasis> based on malondialdehyde,proline content,specific leaf area and carbon isotope discrimination for drought tolerance and divergence of species at DNA level

Cenchrus (family Poaceae) is an important component of major grass covers of the world. Largely it is apomictic and both annual and perennial species exist in nature. Variations in contents of malondialdehyde, proline, specific leaf area and carbon isotope discrimination for drought tolerance were estimated among eight prominent species of Cenchrus. Simultaneously, genetic variations were also estimated by employing 187 RAPD primers. Of these, 23 primers did not react, 2 performed poorly and 7 produced many non-scorable bands and one primer yielded a single monomorphic band. Rest of the 154 primers generated one or more unambiguously scorable fragments. Twelve hundred and four of the 1,296 putative loci were polymorphic (93%) between at least one pair-wise comparisons among eight species. Dice coefficient and neighbor-joining algorithm analyses showed clustering patterns that fit with the known habitat of the species except perennial, C. myosuroides which formed a node between two annuals species. When these species were subjected to water stress tolerance test, a correlation (r = 0.612) between specific leaf area (SLA) and carbon isotope discrimination (CID) and difference in levels of drought tolerance based parameters among eight species were observed. Of the eight species investigated two annuals viz., C. biflorus and C. echinatus showed highest level of genetic similarity which was also evident from the similar levels of SLA, MDA, proline contents and carbon isotope discrimination values observed in these two species.     

PCR primed with minisatellite core sequences yields species-specific patterns and assessment of population variability in fishes of the genus Brycon

Minisatellite core sequences were used as single primers in polymerase chain reaction (PCR) to amplify genomic DNA in a way similar to the random amplified polymorphic DNA methodology. This technique, known as Directed Amplification of Minisatellite‐region DNA, was applied in order to differentiate three neotropical fish species (Brycon orbignyanus, B. microlepis and B. lundii) and to detect possible genetic variations among samples of the threatened species, B. lundii, collected in two regions with distinct environmental conditions in the area of influence of a hydroelectric dam. Most primers generated species‐specific banding patterns and high levels of intraspecific polymorphism. The genetic variation observed between the two sampling regions of B. lundii was also high enough to suggest the presence of distinct stocks of this species along the same river basin. The results demonstrated that minisatellite core sequences are potentially useful as single primers in PCR to assist in species and population identification. The observed genetic stock differentiation in B. lundii associated with ecological and demographic data constitute a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity of this endangered fish species.