使用RAPD分析辨别印度半岛的五种结鱼

用RAPD分析研究了结鱼种组(黄鳍结鱼Tor putitora,结鱼Tor tor,库德里结鱼Tor khudree,Tor mosalmahanadicus和墨脱四■魮Neolissochilus hexagonolepis)5个物种的遗传关系。在所测试的69个随机引物中,11个引物能够在所有5个物种中扩增出稳定的条带。RAPD带型显示,综合使用这些RAPD标记能够区分这5个物种,但Tor mosal mahanadicus和黄鳍结鱼享有相似的带型。UPGMA分析揭示出3个独特的分支,第一支由黄鳍结鱼、Tor mosal mahanadicus和结鱼组成,第二支是库德里结鱼,第三支是墨脱四■魮。Tor mosal mahanadicus的分类地位在不同学者间存在分歧,被认为是库德里结鱼或结鱼的亚种,但我们的结果表明,相对而言,Mahanadi河中的Tor mosal mahanadicus与黄鳍结鱼的进化关系更近,因此有必要对其系统分类地位进行重新评估。     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

Molecular characterization of eight Indian Snakehead species (Pisces: Perciformes Channidae) using RAPD markers

Murrels (Perciformes; Channidei; Channidae) are unique group of freshwater air breathing fishes having a confined distribution to African and Asian continents. The phylogenetic relationship among eight Channid species viz. Channa aurantimaculata, Channa bleheri, Channa diplogramma, Channa gachua, Channa marulius, Channa punctatus, Channa stewartii and Channa striatus were investigated using RAPD markers. Eight random oligodecamers viz. OPAC03, OPAC05, OPAC07, OPAC09, OPAC19, OPA10, OPA11 and OPA16 were used to generate the RAPD profile. Estimates of Nei’s (Genetics, 89:583–590, 1978) unbiased genetic distance (D) demonstrated sufficient genetic divergence to discriminate the samples of different species and the values ranged from 0.3292 to 0.800 The present RAPD analyses strongly substantiate the view of earlier morphological and osteological studies of Channid species, the closer association among species in “gachua” and “marulius” groups.     

Phylogeny analysis of 25 apple rootstocks using RAPD markers and tactical gene tagging

RAPD (random amplified polymorphic DNA) markers were used to fingerprint eight commercially available apple rootstocks (Nertchinsk, Northern Spy, Osman, Heyer 12, M.1, M.9, M.26 and MM.106), 10 winter hardy offsprings derived from the cross of Nertchinsk x M.9, six winter hardy offsprings derived from the cross of Nertchinsk x M.26 and one winter hardy offspring derived from each of the two crosses between Osman x Heyer 12 and Northern Spy x M.1. Phylogeny analysis using parsimony allowed us to draw the genetic relationship between these lines using only RAPD markers data. The resulting cladogram was compared to the true genetic relationship between these lines in order to assess the efficiency of RAPD markers in determining accurately the phylogenetic relationship. We also developed a DNA fingerprinting system based on 13 informative RAPD loci amplified by five RAPD primers that allowed the rapid identification of apple rootstocks.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

用RAPD标记研究蚱属五个种间的亲缘关系

用RAPD技术对蚱属5种蚱基因组DNA的多态性进行研究。在事先优化的反应条件下用12个随机引物扩增, 共得到84条清晰稳定的多态性片段,片段长度为200～2 000 bp。统计这些片段,根据扩增片段的共享度计算出相对遗传距离指数,然后用UPGMA和NJ聚类方法对其进行分析,构建系统树,确定了它们相互间的亲缘关系。     

Molecular identification and phylogenetic relationships of seven Indian Sciaenids (Pisces: Perciformes,Sciaenidae) based on 16S rRNA and cytochrome c oxidase subunit I mitochondrial genes

The partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) mitochondrial genes were analyzed for species identification and phylogenetic relationships among the commercially important Indian sciaenids (Otolithes cuvieri, Otolithes ruber, Johnius dussumieri, Johnius elongatus, Johnieops vogleri, Otolithoides biauritus and Protonibea diacanthus). Sequence analysis of both genes revealed that the seven species fell into three distinct groups, which were genetically distant from each other and exhibited identical phylogenetic resolution. Partial sequences of both the genes provided sufficient phylogenetic information to distinguish the seven sciaenids indicating the usefulness of mtDNA-based approach in species identification.     

Molecular characterization of some Indian Aloe vera populations through RAPD and ITS markers

The most important and well-known medicinal plant among ~400 species of the genus Aloe is Aloe vera. It is widely used in pharmaceutical, cosmetic, and food industries. Identification and assessment of genetic relationship among the populations and cultivars is needed for conservation and sustainable utilization of this commercially important plant. DNA fingerprinting with random amplified polymorphic DNA (RAPD) marker and Internal Transcribed Spacer (ITS1 and ITS2) of ribosomal DNA sequence analysis were carried out to assess the genetic diversity among populations of Aloe vera collected from geographically different four districts of West Bengal and Jodhpur, Rajasthan. RAPD profiles yielded 158 amplicons showing ~87.34% polymorphism. Analyses of ITS sequences showed that in contrast to ITS2, the length and %GC content (53.6–77.3%) of ITS1 varied within populations. Multiple sequence alignment data reveal that substitutions, insertions, and deletions have arisen at various positions in the ITS regions suggesting polymorphism. A 5′-GGCGCGATGGGCGCCAAGGAA-3′ sequence in ITS1 is conserved in all populations, except AvS4. RAPD dendrogram and topologies of the NJ, Parsimony and ML tree generated from ITS1 sequence revealed that there is a close genetic similarity among AvS1, AvS4, and AvS7 populations. These genetic studies may contribute to plant improvement programs of A. vera.     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

利用RAPD分子标记对番茄杂交种纯度的鉴定研究

应用RAPD(RandomlyamplifiedpolymorphicDNA)分子标记对番茄京丹1号和毛粉802的F1代杂交种纯度进行鉴定的实验研究。该项研究使用了10个碱基的单随机引物和10个碱基的双随机引物进行扩增。在60个单引物扩增反应中获得7个京丹1号父本特有的核酸标记片段。但在14个双随机引物对京丹1号和毛粉802杂交组合的扩增反应中获得了7个京丹1号F1代杂交种特有的核酸标记片段和5个毛粉802父本特有的标记带。实验结果显示,双引物的扩增反应对鉴定双亲亲缘关系极近的杂交种纯度较单引物扩增反应更有效。其中,京丹1号的14个标记片段在北京蔬菜研究中心,种子纯度检测室又进行了重复扩增实验。实验结果为87%的RAPD标记可以在使用不同的PCR仪和不同来源的Taq酶的实验条件下得到。RAPD分子标记技术对鉴定双亲亲缘关系极近的杂交种纯度是真实可靠的。     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Molecular identification of Chinese cultivated Porphyra (Bangiaceae, Rhodophyta) based on the rDNA internal transcribed spacer-1 sequence and random amplified polymorphic DNA markers

Porphyra yezoensis Ueda and P. tenera Kjellman are two common commercial seaweeds cultivated in China, but it is difficult to identify them routinely because they are morphologically and systematically very close. The nuclear ribosomal internal transcribed spacer-1 (ITS-1) region of nine selected Porphyra cultivars from China were sequenced and determined. Combined with some ITS-1 data from GenBank, the phylogenetic analysis inferred from the neighbour-joining method indicated that the Porphyra species in this study exhibited clear taxonomic relationships and could be identified clearly to species. Based on the results, the random amplified polymorphic DNA (RAPD) technique was applied to distinguish 12 Porphyra cultivars, some of which turn out to be different lineages of the same Porphyra species. Eight specific RAPD markers were scored and used to construct a fingerprint that could distinctly identify different Porphyra cultivars. The results suggest that both the rDNA ITS-1 sequence and RAPD markers are useful methods to identify Porphyra cultivars, and may also be valid for phylogenetic and taxonomic studies.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Identification of white truffle species using RAPD markers

Morphologically very similar species of white truffle were analyzed by the random amplified polymorphic DNA (RAPD) technique. Species-specific RAPD fragments were selected and pairs of primers were designed on their sequences. Sequence-characterized amplified regions were developed and applied to identify these species throughout their entire life cycle: fruit body, mycelium, ectomycorrhiza. This procedure provides an unambiguous and rapid tool for typing species whose morphology is very similar.     

Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers

Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population. Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique ∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male and female samples of four cultivars. The unique ∼1,400 bp fragment was present in male individuals of all the four cultivars and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain the sex of jojoba plants at an early stage of development of the taxon.     

Molecular identification and phylogenetic relationship of green algae,Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers

Spirogyra is found in a wide range of habitats, including small stagnant water bodies, rivers, and streams. Spirogyra ellipsospora is common in northern Thailand. Species identification of the Spirogyra species based only on morphological characteristics can be difficult. A reliable and accurate method is required to evaluate genetic variations. This study aims to apply molecular approaches for the identification of S. ellipsospora using microsatellites and rbcL markers. Based on DNA sequencing, the rbcL gene was sequenced and the data was analyzed using the BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. The sequence of S. ellipsospora from this study revealed definitive identity matches in the range of 99% for the consensus sequences of S. ellipsospora. The 10 primers of ISSR could be amplified by 92 amplification fragments. The DNA fragments and the rbcL sequence data grouped the Spirogyra specimens into two distinct clusters.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.