Genetic analysis of east and west coast populations of Penaeus monodon from India based on random amplified polymorphic DNA

The east and west coast populations of wild Penaeus monodon in India were genetically characterized by RAPD analysis using six highly polymorphic primers reported earlier. The average genetic similarities within populations, based on profiles generated by all the six primers, were 0.828 and 0.851 for the east and west coast populations, respectively, values with individual primers ranging from 0.744 to 0.889. The average genetic similarity between populations across all the primers was 0.774. The number of bands found to be polymorphic were 38 (51.35%) and 37 (50.68%) in the east and west coast populations, respectively. Primer 5 yielded the highest level of polymorphism (63.63%) in the east coast population whereas primer 3 yielded the lowest level of polymorphism (36.36%) in the west coast population. The study reveals the existence of genetic variation in P. monodon stocks providing scope for genetic improvement through selective breeding. It also provides baseline data for future work on population structure analysis of P. monodon.

Population genetic structure of Carchesium polypinum (Ciliophora : Peritrichia) in four chinese lakes inferred from ISSR fingerprinting: High diversity but low differentiation

Although the peritrichous ciliate Carchesium polypinum is common in freshwater, its population genetic structure is largely unknown. We used inter-simple sequence repeat (ISSR) fingerprinting to analyze the genetic structure of 48 different isolates of the species from four lakes in Wuhan, central China. Using eight polymorphic primers, 81 discernible DNA fragments were detected, among which 76 (93.83%) were polymorphic, indicating high genetic diversity at the isolate level. Further, Nei's gene diversity (h) and Shannon's Information index (I) between the different isolates both revealed a remarkable genetic diversity, higher than previously indicated by their morphology. At the same time, substantial gene flow was found. So the main factors responsible for the high level of diversity within populations are probably due to conjugation (sexual reproduction) and wide distribution of swarmers. Analysis of molecular variance (AMOVA) showed that there was low genetic differentiation among the four populations probably due to common ancestry and flooding events. The cluster analysis and principal component analysis (PCA) suggested that genotypes isolated from the same lake displayed a higher genetic similarity than those from different lakes. Both analyses separated C. polypinum isolates into subgroups according to the geographical locations. However, there is only a weak positive correlation between the genetic distance and geographical distance, suggesting a minor effect of geographical distance on the distribution of genetic diversity between populations of C. polypinum at the local level. In conclusion, our studies clearly demonstrated that a single morphospecies may harbor high levels of genetic diversity, and that the degree of resolution offered by morphology as a marker for measuring distribution patterns of genetically distinct entities is too low.

青藏高原东缘青杨（Populus cathayana Rehd.）遗传多样性研究

青杨（Populus cathayana Rehd.）是青杨派杨树的主要树种之一，为我国特有乡土树种，其主要分布区之一是我国的青藏高原，集中分布地带在甘肃省中部及青海省东部，四川省西北部岷江上游和松潘等地区。本研究以青藏高原东缘青杨天然分布区的6个群体143个个体为材料，用AFLP、SSR和叶绿体SSR分子标记分析青杨天然群体的遗传多样性，分析其遗传结构和分化，比较6个群体间遗传多样性的高低和群体间的遗传关系。旨在为青杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据。通过以上研究，得出如下主要研究结果： 1 AFLP分子标记研究结果 采用4对选择性引物对6个青杨天然群体143个个体进行分析，扩增谱带分析共检测到175个位点，其中173个位点表现为多态，多态位点百分率高达98.9%。从整体上表现出较高的遗传多样性，Nei’s基因多样度(h)水平为0.306。从青杨天然群体位点分布来看，有高达20%的位点（32位点）为群体所特有，仅有9.14%的位点（16位点）在所有群体中存在。群体间的遗传分化极大，所有遗传变异中，有48.9%的遗传变异存在于群体间。在个体群丛（Individuals cluster）和主坐标（PCO analysis）分析中，青杨各群体未呈现任何地理模式，Mantel检测也显示各群体间遗传距离与地理距离无明显相关。研究认为，由于地理和空间上大尺度的隔离和地形地貌复杂使得群体间无法进行基因交流，导致群体间遗传分化极大，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 2 SSR分子标记研究结果 在SSR分析中，7个位点在6个青杨天然群体143个个体中共检测到79个等位基因，每位点检测到的等位基因数在5-16之间，平均11.3个，总体上多态位点百分率达100%。平均观察杂合度和期望杂合度分别为0.792和0.802。Hardy-Weinberg平衡检验表明青杨大部分群体都处于非平衡状态，群体大部分位点都是偏离哈迪-温伯格平衡（76.3%），只有23.7%的测验满足哈迪-温伯格平衡。分析青杨天然群体内和群体间的遗传变异，基因分化系数（GST）为0.373，即有62.7%的遗传变异存在群体内，37.3%的遗传变异存在群体间。群体内的遗传变异高于群体间水平。根据各群体遗传距离UPGMA聚类分析，有来自相临分布区、近似气候类型的群体聚在一起的趋势，但Mantel检测反映遗传距离与地理距离间并无明显相关性。 3 cpSSR分子标记研究结果 分析来自青藏高原东缘6个青杨天然群体，所用cpSSR引物中有5对cpSSR引物（CCMP2、CCMP5、SCUO01、SCU03、SCU07）都表现较高的多态性，单个引物检测的片段数都在4以上。5对cpSSR引物共检测片段数26个，组成了12种叶绿体DNA单倍型。各群体的单倍型分布和频率有较大差异，群体单倍型多样性范围为0-0.4926，TS、JZ、PW和SHY群体单倍型多样性高于QHY和LED群体水平。本研究发现，分布在青藏高原东缘的青杨天然群体，群体间不存在共享的单倍型，各群体间存在极大的遗传分化（GST=0.9223）。从青藏高原东缘地区经历的地质历史事件来看，第四纪的冰期气候变迁可能是造成青杨现今遗传结构模式的主要因素之一。根据单倍型在各群体的分布情况，进行青杨群体聚类分析结果，各群体无明显的分组现象，青杨各群体也未呈现任何清晰地理模式。 由于不同分子标记在对群体遗传多样性检测能力与效率上存在差异，所以三种标记检测的青藏高原东缘青杨天然群体遗传多性水平也不尽一致，但在与用同种方法检测其它物种或同一物种不同种源群体比较，三种分子标记方法都揭示了青藏高原东缘青杨天然群体具有中等偏上的遗传多样性水平。结果分析表明，群体间遗传分化极大，这是由于青杨天然群体分布于青藏高原东缘，既有高原又有高山峡谷，由于地理和空间上大尺度的隔离和地形地貌复杂导致了基因流物理上的阻隔。三种分子标记研究结果经Mantel分析检测，遗传距离与地理距离之间都无明显相关性。较为一致的解释是，青杨分布区域地理和空间上大尺度的隔离和和地形地貌复杂导致群体之间不存在均匀扩散现象，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 The wide geographical and climatic distribution of P. cathayana Rehd. indicates that there is a large amount of genetic diversity available, which can be exploited for conservation, breeding programs and afforestation schemes. The results are as follows: 1 Research results of AFLP genetic diversity In present study, genetic diversity was evaluated in the natural populations of P. cathayana originating from southern and eastern edge of the Qinghai-Tibetan Plateau of China by means of AFLP markers. For four primer combinations, a total of 175 bands were obtained, of which 173 (98.9%) were polymorphic. Six natural populations of P. cathayana possessed different levels of genetic diversity, high level of genetic differentiation existed among populations (GST=0.489) of P. cathayana. Individuals cluster and PCO analysis based on Jaccard’s similarity coefficient also showed evident population genetic structure with high level population genetic differentiation. The long evolutionary process coupled with genetic drift within populations, rather than contemporary gene flow, are the major forces shaping genetic structure of P. cathayana populations. Moreover, there is no correspondence between geographical and genetic distances in the populations of P. cathayana, seldom gene exchange among populations and different selection pressures may be the causes. Our finding of different levels of genetic diversity within population and high level of genetic differentiation among populations provided promising condition for further breeding or conservation programs. 2 Research results of SSR genetic diversity In this study, the genetic diversity of P. cathayana was investigated using microsatellite markers. In a total of 150 individuals collected from six natural populations in the southeastern part of the Qinghai-Tibetan Plateau in China, a high level of microsatellite polymorphism was detected. At the seven investigated microsatellite loci, the number of alleles per locus ranged from 5 to 16, with a mean of 11.3, the observed heterozygosities across populations ranged from 0.408 to 0.986, with a mean of 0.792, and the expected heterozygosities across populations ranged from 0.511 to 0.891, with a mean of 0.802. The proportion of genetic differentiation among populations accounted for 37.3% of the whole genetic diversity. The presence of such a high level of genetic diversity could be attributed to the features of the species and the habitats where the sampled populations occur: The southeastern part of the Qinghai-Tibetan Plateau is regarded as the natural distribution and variation center of the genus Populus in China. Variation in environmental conditions and selection pressures in different populations, and topographic dispersal barriers could be factors associated with the high level of genetic differentiation found among populations. The populations possessed significant heterozygosity excesses, which may be due to extensive population mixing at the local scale. The cluster analysis showed that the populations are not strictly grouped according to their geographic distances but the habitat characteristics also influence the divergence pattern. In addition, we suggest that population SHY should be regarded as an ecologically divergent species of P. cathayana. 3 Research results of cpSSR genetic diversity Genetic diversity of six natural populations of P. cathayana originating from the southeastern part of the Qinghai-Tibetan Plateau in China was studied by use of cpSSR markers. Based on 5 pairs of polymorphic primers screened from 12 pairs of primers, twenty-six different length fragments and twelve different kinds of haplotypes were reduced in 143 samples. There were significant variant haplotypes among the populations．There were no shared haplotypes found among populations, analysis of molecular variance indicated that a high proportion of the total genetic variance was attributable to variations among populations (92.23%). The pattern of genetic structure which is associated with spatial separation, variation in environmental conditions and selection pressures in different populations, is also the result of geological historical factor. A molecular phylogenetic tree based on the 12 haplotypes showed that the populations are not strictly grouped according to their geographic distances.

Estrutura genética de populações de Trichilia pallida Swartz (Meliaceae) por marcadores RAPD

Trichilia pallida Swartz is an Atlantic Forest shady climax tree of Meliaceae family that presents insecticide properties against chewing insects like as some family trees, making it interesting for forestry uses. Forty plants of Oito Pontas Farm population were collected in Bofete County, Santa Genebra Ecological Station in Campinas County, and Caetetus Ecological Station in Gélia County, all in the Sao Paulo State, Brazil. Leaf DNA analysis was used by RAPD method, that showed 10 highly polymorphic primers, with 72 dominant markers, used to estimate genetic diversity within and among populations. The polymorphism within populations varied from 90.3 to 97.2%, and the effective allele number varied from 1.46 ± 0.33 to 1.57 ± 0.33, while the average of genetic differentiation of populations varied from 0.27 ± 0.18 to 0, 33 ± 0.15. The gene diversity in the total population (H T) was 0.334 ± 0.02, while the average gene diversity within populations (H s) was 0.292 ± 0.017, and the coefficient of gene differentiation (G ST) was 0.125, Bofete and Campinas populations had the smallest Nei's genetic distance (0.049) and the distances of both with Gália were 0.117 and 0.107, respectively.

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

Polymorphic microsatellite loci for the zebra shark Stegostoma fasciatum

We report on the development and characterization of 14 polymorphic microsatellite loci in the zebra shark (Stegostoma fasciatum). Five tetranucleotide and nine dinucleotide loci were polymorphic with heterozygosities ranging from 0.400 to 0.967 and from three to 22 alleles per locus. Cross-species amplification of these zebra shark primers on four other species of orectolobid sharks was not successful.

An identification tool for the Australian weedy Sporobolus species based on random amplified polymorphic DNA (RAPD) profiles

Based on morphological features alone, there is considerable difficulty in identifying the 5 most economically damaging weed species of Sporobolus [viz. S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur and Schinz, S. fertilis (Steud.) Clayton, S. africanus (Poir.) Robyns and Tourney, and S. jacquemontii Kunth.] found in Australia. A polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) technique was used to create a series of genetic markers that could positively identify the 5 major weeds from the other less damaging weedy and native Sporobolus species. In the initial RAPD profiling experiment, using arbitrarily selected primers and involving 12 species of Sporobolus, 12 genetic markers were found that, when used in combination, could consistently identify the 5 weedy species from all others. Of these 12 markers, the most diagnostic were UBC51490 for S. pyramidalis and S. natalensis; UBC43310.2000.2100 for S. fertilis and S. africanus; and ORA20850 and UBC43470 for S. jacquemontii. Species-specific markers could be found only for S. jacquemontii. In an effort to understand why there was difficulty in obtaining species-specific markers for some of the weedy species, a RAPD data matrix was created using 40 RAPD products. These 40 products amplified by 6 random primers from 45 individuals belonging to 12 species, were then subjected to numerical taxonomy and multivariate system (NTSYS pc version 1.70) analysis. The RAPD similarity matrix generated from the analysis indicated that S. pyramidalis was genetically more similar to S. natalensis than to other species of the 'S. indicus complex'. Similarly, S. jacquemontii was more similar to S. pyramidalis, and S. fertilis was more similar to S. africanus than to other species of the complex. Sporobolus pyramidalis, S. jacquemontii, S. africanus, and S. creber exhibited a low within-species genetic diversity, whereas high genetic diversity was observed within S. natalensis, S. fertilis, S. sessilis, S. elongates, and S. laxus. Cluster analysis placed all of the introduced species (major and minor weedy species) into one major cluster, with S. pyramidalis and S. natalensis in one distinct subcluster and S. fertilis and S. africanus in another. The native species formed separate clusters in the phenograms. The close genetic similarity of S. pyramidalis to S. natalensis, and S. fertilis to S. africanus may explain the difficulty in obtaining RAPD species-specific markers. The importance of these results will be within the Australian dairy and beef industries and will aid in the development of integrated management strategy for these weeds.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Development of type I genetic markers from expressed sequence tags in highly polymorphic species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: - 1) priming site polymorphism in the template leads to inferior or erratic amplification; - 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions, and; - 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.

Isolation and characterization of 18 polymorphic microsatellite markers in Chinese mandarin fish Siniperca chuatsi (Basilewsky)

Eighteen microsatellite markers were isolated and characterized using an enrichment protocol in the Chinese mandarin fish Siniperca chuatsi (Basilewsky), a commercially important piscivorous fish in China. Out of 48 pairs of primers designed, 18 loci exhibited polymorphism with three to six alleles (mean 4.4 alleles/locus) and average observed heterozygosity ranged from 0.633 to 0.833 (mean 0.748) in a test population from Dongting Lake of China. Except for two loci, all other 16 loci were in the Hardy-Weinberg equilibrium. These markers would be useful for such studies as population genetics, ecology and selective breeding of the Chinese mandarin fish in future.

Development of polymorphic EST markers in Penaeus monodon: applications in penaeid genetics

The objective of this study was to develop type I markers for genome mapping and other genetic studies of Penaeus monodon. Primers were designed based on expressed sequence tags (ESTs) from a P monodon cephalothorax cDNA library to amplify 100-300 bp products. 34 of the primer pairs successfully amplified PCR products from genomic DNA. Single-strand conformation polymorphism analysis showed that similar to 30% of the ESTs tested exhibit polymorphism in a test panel of P monodon individuals. Mendelian inheritance of the EST-derived markers has been established in two international reference mapping families of P monodon, and mapping of these markers is in progress. Some ESTs were successfully amplified from other Penaeus species (P. chinensis, P japonicus and P vannamei), indicating that the markers are applicable in cross-species comparison. Two populations of P. japonicus could be differentiated using one of the ESTS. In conclusion, the polymorphic EST markers developed in this study are applicable in genome mapping and population genetic studies of penaeid shrimp. (C) 2002 Elsevier Science B.V. All rights reserved.

Nine microsatellite DNA primers for Hippophae rhamnoides ssp sinensis (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the and regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.

The application of randomly amplified polymorphic DNA (RAPD's) to the study of Lasaea rubra (Montagu).

This paper describes the random amplification of polymorphic DNA markers (RAPDs) in Lasaea rubra (Erycinidae: Bivalvia). Present evidence suggests that L. rubra is an asexual species; however, the exact mode of clonal reproduction in this species is still a matter of debate. In this preliminary study, four of the primers used generated polymorphic RAPDs. One primer was able to distinguish between individuals from the same or different crevice population. This same primer also resolved a single band difference between otherwise identical RAPD patterns of a parent and its offspring. No familial differences have been detected in several previous studies using allozyme electrophoresis. This paper suggests that many polymorphic markers could be obtained with this species using the RAPD technique. Population genetic analysis of L. rubra has long been hampered by a dearth of polymorphic markers due to its small size. These findings suggest that this technique has the potential to further the study of population genetics in this asexual species.