Transferability of STS markers in studying genetic relationships of marvel grass (Dichanthium annulatum)

Transferability of sequence-tagged-sites (STS) markers was assessed for genetic relationships study among accessions of marvel grass (Dichanthium annulatum Forsk.). In total, 17 STS primers of Stylosanthes origin were tested for their reactivity with thirty accessions of Dichanthium annulatum. Of these, 14 (82.4%) reacted and a total 106 (84 polymorphic) bands were scored. The number of bands generated by individual primer pairs ranged from 4 to 11 with an average of 7.57 bands, whereas polymorphic bands ranged from 4 to 9 with an average of 6.0 bands accounts to an average polymorphism of 80.1%. Polymorphic information content (PIC) ranged from 0.222 to 0.499 and marker index (MI) from 1.33 to 4.49. Utilizing Dice coefficient of genetic similarity dendrogram was generated through un-weighted pairgroup method with arithmetic mean (UPGMA) algorithm. Further, clustering through sequential agglomerative hierarchical and nested (SAHN) method resulted three main clusters constituted all accessions except IGBANG-D-2. Though there was intermixing of few accessions of one agro-climatic region to another, largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 scale also showed large number of nodes (11 to 17) having strong clustering (> 50). Thus, results demonstrate the utility of STS markers of Stylosanthes in studying the genetic relationships among accessions of Dichanthium.     

柚类资源及其近缘种SSR标记的分子评价

采用31对SSR引物对122份柚类种质资源及其近缘种的遗传多样性进行了研究.31对SSR引物可以检测到335个等位基因变异,平均每个位点可检测到9.85个等位基因.每个位点多态性信息指数(PIC)平均值为0.7085.从这些引物中有效地筛选出21对高信息量SSR引物,与31对引物在122份柚类资源间遗传关系达到极显著相关(r=0.99104).柚类资源及其近缘种等位基因平均数(A)、平均杂合位点百分比(P)、SSR表型杂合度(H0)分别为2.8、83.54%、0.524.用UPGMA方法将122份研究材料分成7个组群,110个柚类品种在相似系数0.712时可细分成18个亚组,分类结果有利于今后有目的地利用这些丰富育种材料的遗传背景.图3表2参21     

Identification of Tsuga Germplasm by Morphological Characters and RAPD Markers

Germplasm collection is important to preserve and maximize genetic diversity for germplasm conservation. Tsuga dumosa （ D. Don） Eichler in Engler ＆ Prantl. and T. chinensis var. forrestii （Downie） Silba germplasm was collected from three localities in China： Mt. Yulong, Wenfeng Temple and Mt. Dishiergu, Yunnan Province. Accessions were identified based on morphological characters and RAPD markers. The shapes of the apices and margins of needles were examined, and the length and width of needles, cones and seeds from accessions of mature plants were used to compare the morphological differences and to identify the germplasm. Molecular markers generated by randomly amplified polymorphic DNA （RAPD） were also used to characterize the taxa. Although the clustering based on RAPD markers was inconsistent with the morphological characters of the needles, based on the overall morphological characters and on RAPD markers, the accessions from Mt. Yulong and Wenfeng Temple were identified as T. chinertsis var. forrestii, and those from Mt. Dishiergu identified as T. dumosa. Taxonomic identification of the accessions was made based on morphology and by RAPD markers concurred. The results indicate that the shapes of the apices and margins of needles particularly from young plants could not be used as a possible key to identify T. dumosa and T. chinertsis var. forrestii. Fig 6, Tab 3, Ref24     

Identification of species-specific RAPD markers in genus Cenchrus

Cenchrus is an important component of major grass cover of world. Similar to the other major tropical grasses most of the species in genus Cenchrus are also apomictic in nature hence correct and precise identification of accessions and species are problematic and dubious. In the present study 187 decamer oligonucleotide primers were tested for PCR-based DNA amplification of six prominent species of genus Cenchrus. Of these, 32 potential repetitive and polymorphic primers were tested for identification of species-specific markers for C. ciliaris, C. setigerus, C. pennisetiformis, C. prieurri, C. biflorus and C. myosuroides. These primers yielded 51 unique RAPD markers either specific to a species (37) or shared by two or more species (14). Maximum markers were shared between C. ciliaris and C. setigerus confirming theirmore closeness to each other Primers like OPF09, OPF11, OPR15, OPAJ11, OPQ10 and OPAK20 generated strong intense bands can be used on priority in identifying the species from their natural habitat for the development of species-specific core germplasm. Due to apomictic nature this is the prime method of developing cultivars, as morphological characters are largely unable to distinguish them. The level of variation observed clearly suggest RAPD as an appropriate marker for genetic studies and in identifying the lines with species-specific markers for Cenchrus germplasm management activity and also maintaining identity and purity for proprietary reasons.     

Use of EST database markers from M. truncatula in the transferability to other forage legumes

In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60 degreesC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. Atotal of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNAfrom 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55 degreesC and 32.3% at 60 degreesC annealing temperature. 24 alleles of the total 50 (48%) at 55 degreesC and 27 of 51 (53%) at 60 degreesC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where SequentialAgglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.     

Acclimation of the European sea bass to freshwater: monitoring genetic changes by RAPD polymerase chain reaction to detect DNA polymorphisms

We investigated the use of the polymerase chain reaction (PCR) and the associated random amplification of polymorphic DNA (RAPD) technique to study variation in samples of the sea bass, Dicentrarchus labrax, before and after acclimation to freshwater. Acclimation trials were repeated twice, in 1989 and 1990, for two samples originating from the same broodstock (individuals from different localities along the Italian coasts), with overall mortality rates averaging 94 and 75% in the 2 yr, respectively. Analyses are based on 126 polymorphic RAPD markers, scored in at least 39 individuals for each of the starting and acclimated samples in both years. Analysis of RAPD patterns revealed high levels of DNA polymorphism in both 1989 and 1990 starting samples. The acclimated samples maintained similar polymorphism levels. Shifts in marker frequencies between starting and acclimated samples occurred in both years. A correspondence analysis carried out on multimarker individual profiles suggests that the two starting samples resulted from uneven sampling of the same heterogenous broodstock. This analysis clearly separates RAPD phenotypes from starting and acclimated samples in both years and identifies the RAPD markers responsible for such displacement. Patterns of RAPD variation are compared with previous allozymic studies carried out on the same samples. A major difference between the two studies was the number of markers available. Fewer allozyme loci were studied than were RAPD markers. The cause of repeated shifts for allozyme alleles in replicate experiments were almost certainly due to selection, while statistical chance could explain the repeated shift of only one out of more than 100 RAPD markers. We have shown that RAPD analysis, if carried out carefully, is quite reproducible and sensitive enough to reveal high levels of variation among individuals from the same broodstock. A major drawback of this approach is the still unclear inheritance patterns of RAPD polymorphisms. The use of multivariate analyses is suggested as a possible alternative to traditional population genetics techniques to analyze patterns of variation in the absence of a precise genetic interpretation.     

11个猕猴桃品种间的遗传多样性分析

利用随机扩增多态性DNA标记(RAPD)技术对采自四川省猕猴桃科研基地的11个猕猴桃品种进行了遗传多样性分析.通过引物筛选,从15个RAPD引物中扩增出135条带,其中100条为多态带,占74.07%.UPGMA聚类分析结果揭示了各品种间的亲缘关系.RAPD标记说明猕猴桃品种间遗传距离与地理分布有一定关系,地理位置较近的材料能聚在一起.分子标记可用于猕猴桃种质资源的分类、鉴定及良种选育.图2表2参15     

用SSR标记检测杂交籼稻三系亲本的遗传差异

随机选用分布于水稻(Oryza sativa L.)12条染色体上的110对引物,共筛选出26对具有多态性的SSR引物,对24份杂交籼稻三系亲本材料进行遗传多样性和亲缘关系分析.全部24份亲本中共检测出116对等位基因,每个SSR引物可检测到的等位基因数目为2～7个不等,平均每对引物可以检测到4.4个等位基因.多态信息量(PIC)的变动范围为0.068 9～0.803 6,平均PIC值为0.603 4;期望杂合度(He)的变动范围为0.081 6～0.829 8,平均值为0.616 9;多样性指数(1)变幅为0.173 2～1.791 3,平均为1.160 0.对供试亲本材料根据遗传距离进行UPGMA聚类分析,结果表明,SSR微卫星标记可以将24份亲本材料分为3个大群,各组亲本间的遗传差异较大.通过分子标记辅助育种,可以有效地鉴定供试亲本材料之间的亲缘关系,分析预测新种质材料的遗传差异,可作为选育强优势组合杂交水稻的重要参考指标之一.图2表3参24     

Genetic diversity of blackspot seabream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>) populations off Spanish Coasts: a preliminary study

Genetic diversity among four natural samples of Blackspot seabream (Pagellus bogaraveo, Brünnich, 1768) from different fishing grounds exploited by Spanish fisheries was analyzed through the use of 12 microsatellite markers. The samples were captured off the Spanish coasts from the Mediterranean Sea to the Cantabrian Sea within the same continental slope. High levels of genetic diversity were revealed for every population and every locus was polymorphic at the 0.95 level. The average number of alleles, average heterozygosity and PIC were found to be 15.75, 0.833 and 0.818, respectively. In general, population differentiation was not detected in these samples. Through AMOVA, a low level of variation between regions (Mediterranean vs. Atlantic samples) was observed, though this was not significant. A larger percentage of total variation was observed inside the ‘within populations’ class. Thus, AMOVA did not reveal any significant population substructure. Moreover, no correlation was found between geographical and F_ST estimates and the observed results did not allow the improvement of a model of isolation by distance. The high homogeneity revealed by means of these markers could indicate the absence of physical frontiers between the geographical areas analyzed in this survey, especially between Atlantic and Mediterranean areas.     

Genetic Diversity and Tests of the Hybrid Origin of the Endangered Yellow Larkspur

Abstract: Delphinium luteum ( Ranunculaceae), an endangered larkspur, is restricted to two wild populations near Bodega Bay, California. The total number of individuals in these two populations is estimated to be <100. We used allozyme and random amplified polymorphic DNA ( RAPD) markers to (1) assess levels and patterns of genetic diversity in one wild population and two cultivated populations and (2) test the hypothesis that D. luteum is of hybrid origin between D. decorum and D. nudicaule . These data will be used to aid in developing a management plan to conserve the species. The wild population maintains high levels of genetic diversity. Genetic data indicate that both cultivated populations, especially the north Sonoma population, have several allozymes and RAPD markers not found in the wild population and could be used to establish new populations of D. luteum or to enhance the diversity and size of the wild population. The allozyme data did not reveal any fixed differences between D. decorum and D. nudicaule , although allele frequencies of the putative parental populations differed. At these loci, D. luteum resembled D. nudicaule more than D. decorum  . Many unique RAPD markers distinguish each of the three species. The diagnostic markers from populations of D. nudicaule and D. decorum were not additive in the putative hybrid, and these data indicate that D. luteum is not of recent hybrid origin. Conservation of the yellow larkspur should include strategies that use the cultivated populations of D. luteum , but hybridizing D. decorum and D. nudicaule to "recreate" D. luteum is not recommended.     

Analysis of genetic structure of blacklip abalone (Haliotis rubra) populations using RAPD, minisatellite and microsatellite markers

 We investigated the utility of three polymerase chain-reaction (PCR)-based DNA molecular markers in analysing genetic structure of the populations of the blacklip abalone Haliotis rubra (Leach) of Victoria, Australia. The DNA markers included 84 randomly amplified polymorphic DNA (RAPD) bands amplified using six random primers, two minisatellites, GHR (putative growth-hormone-gene-repeat) and MIPR (putative mollusca-insulin-like peptide-gene-repeat), and three microsatellites, RUBGT1 [containing (GT)_n repeats], RUBCA1 [containing (CA)_n repeats] and RUBGACA1 [containing (GACA)_n repeats]. All three types of DNA markers revealed significant subdivision in the H. rubra populations along the coastline. This is postulated as being related to the abalone's relatively short pelagic period and limited dispersion. Further analysis revealed that a Point Cook population sampled from within the semi-enclosed Port Phillip Bay was distinct from two other central zone populations (Apollo Bay and Cape Schanck). The genotypes of microsatellites indicated excessive homozygotes across all the populations at all three microsatellite loci, and possible causes such as larval recruitment pattern and asynchronous spawning are discussed. The excessive homozygotes recorded for the three microsatellite loci contrast with those observed in the minisatellite loci GHR and MIPR, the heterozygosities of which were at Hardy–Weinberg equilibrium. Received: 17 March 1999 / Accepted: 24 November 1999     

RAPD markers suggest genotypic effects on forager specialization in a eusocial wasp

Genetic variability within insect societies may provide a mechanism for increasing behavioral diversity among workers, thereby augmenting colony efficiency or flexibility. In order to assess the possibility that division of labor has a genetic component in the eusocial wasp Polybia aequatorialis, I asked whether the genotypes of workers within colonies correlated with behavioral specialization. Workers specialized by foraging for one of the four materials (wood pulp, insect prey, nectar, or water) gathered by their colonies. I collected foragers on 2 days from each of three colonies and identified the material the foragers were carrying when collected. I produced random amplified polymorphic DNA (RAPD) markers from the genomic DNA of these foragers and estimated genotypic similarity of foragers based on sharing of variable RAPD marker bands. Contingency tests on 20 variable loci per colony showed statistically significant (P <0.05) biases in RAPD marker frequencies among forager types in the three colonies. Patterns of association of RAPD marker bands with specializations were constant in two colonies, but changed between collection days in one colony. RAPD marker biases suggest that division of labor among workers includes a genetic component in P. aequatorialis. Colony-level selection on variation in division of labor is a possible factor favoring the evolutionary maintenance of high genotypic variability (low relatedness) in epiponine wasp colonies and in other eusocial insects. Received: 18 July 1995/Accepted after revision: 1 October 1995     

Genetic similarity between two morphologically similar species of polychaetes

Glycera dibranchiata Ehlers and G. americana Leidy are two closely related and similar species of polychaetes. Electrophoretic studies of 11 enzyme systems encoded by 15 loci reveal that both species are polymorphic at about 50% of their enzyme loci, but that G. dibranchiata is the more variable as judged by heterozygosity per individual, genetic diversity, and effective number of alleles per locus. Both species show marked heterozygote deficiencies at nearly all polymorphic loci, possibly as the result of life-history features. Genetic similarity between the Glycera species suggests divergence at a level between species and sibling species compared to the Drosophila willistoni complex.     

Application of random amplified polymorphic DNA (RAPD) markers to evaluate intraspecific genetic variation in red mullet (Mullus barbatus)

The random amplified polymorphic DNA (RAPD) technique was used to evaluate genetic affinities among eight red mullet (Mullus barbatus L., 1758) samples from the Mediterranean Sea. Twenty-nine random primers were used. Despite the variability which was found within samples, no specific RAPD marker for the discrimination of the populations was detected. The data analysis revealed that the genetic diversity among populations is positively related to their geographic distances. The results of this study indicate that the estimated intraspecific variation may be more␣pronounced with RAPD markers than with allozymes when the two approaches are applied on the same populations. Received: 7 November 1997 / Accepted: 30 May 1998     

利用RAPD技术分析兰属（Cymbidium）品种间的亲缘关系

本文利用RAPD技术来检测兰属13个品种间的亲缘关系。通过对55种10个碱基随机引物的筛选,其中4种引物能得到重复性、稳定性较高扩增产物,四种引物共扩增出93条多态性带,多态率为100％,根据RAPD标记进行邻体聚类分析,表明兰属各种间的亲缘关系与形态学分类结果不完全一致。实验结果认为RAPD技术可用于兰属植物的系统学研究。图2表3参13     

Genetic diversity in some perennial plant species with-in short distances

Distinct morphophysiological variations observed for over 2 years with-in short distances among four perennial plants indicated genetic diversity among the lines growing at three places. The isozyme and SDS polyacrylamide gel banding patterns as genetic markers were used to investigate four perennial species, namely Dalbergia sissoo Roxb., Delonix regia (Boj.) Refin., Cassia fistula L. and Calotropis procera R. Br. Plant materials collected from three locations (Agra, Gwalior and Lucknow) differing in climo-edaphic variables were examined for 4 enzyme systems, viz., esterase, polyphenol oxidase, peroxidase and superoxide dismutase (EST, PPO, PRX and SOD). Among the four isozymes SOD and PRX revealed best discriminating power. Protein banding patterns as well as zymogram revealed that Dalbergia sissoo growing at Gwalior was closer to that of Agra; Delonix regia depicted highest similarity between Lucknow and Agra and Calotropis procera of Lucknow location was more closer to Gwalior than Agra. The results confirm genetic diversity in the species as a means of adaptation to differing climo-edaphic variables.     

麻疯树种质资源遗传多样性的ISSR分析

采用ISSR分子标记技术对大戟科属麻疯树9个自然居群的遗传多样性水平和遗传结构进行了研究.用10个引物对9个居群共135个样品进行了扩增,共获得169条清晰的扩增位点,其中多态性位点164个.POPGENE分析结果表明,麻疯树居群具有丰富的遗传多样水平(多态百分率PPB=97.04%,Neis遗传多样性H=0.2357,Shannon信息指数I=0.3760).AMOVA分析表明,9个自然居群间遗传多样性出现了较大的遗传分化(Gst=0.5398)可能与其有限的基因流(Nm=0.4667)和遗传漂变有关;而居群内有限的遗传分化可能是由于麻疯树自交、近交占主导方式的繁育方式有关.并进行了麻疯树居群的聚类分析.图3表4参19     

Genetic differentiation of populations of the planktonic copepod Labidocera aestiva

Planktonic populations of the calanoid copepod Labidocera aestiva show significant biochemical genetic heterogeneity along the Atlantic coast of the USA. In summer, 1981, copepods were collected by surface tows at Beaufort Inlet, North Carolina; Fort Pierce Inlet, Florida; and Vineyard Sound, Massachusetts. Genetic variation within each population and genetic differentiation among the three populations were studied by micro-acrylamide gel electrophoresis of six loci encoding four enzymes. All six enzyme loci were polymorphic when all populations were considered together, but the North Carolina population was monomorphic at two loci. High genetic variability was indicated by the following: (1) the number of alleles per locus averaged over all loci was 2.57±0.26 SD; (2) the average proportion of loci for which the frequency of the most common allele was not greater than 0.95 was 0.78±0.10; (3) the frequency of heterozygous individuals was 0.25±0.07. Genetic differentiation among population samples in the three regions was demonstrated in several ways: allele frequencies at one aminopeptidase-I locus, Lap-1, differed significantly among samples of the three populations, and there were unique alleles of high frequency at this locus in two population samples. Values of the statistic of genetic distance (D) averaged 0.20±0.08 for pairwise comparisons between all samples. Compared to expected heterozygosity if individuals across the entire range sampled mated at random, there were highly significant heterozygote deficiencies at five of the six loci. Genetic differentiation of populations of L. aestiva may result from (1) differential selection on populations in the three regions, or (2) restricted gene flow between the populations. Gene flow may be limited by geographic separation or differences in life history, such as seasonal presence in the plankton and diapause egg production.Contribution No. 5810 of Woods Hole Oceanographic Institution     

基于RAPD技术检测不同烃链长度咪唑基离子液体对日本三角涡虫的遗传毒性

RAPD标记通常用于遗传作图、分类和系统发育研究,该技术也可用于检测DNA损伤或突变。本研究利用RAPD技术检测了4种不同烃链长度的咪唑基离子液体对日本三角涡虫(Dugesia japonica)的遗传毒性。在20条随机引物中共筛选出11条用于RAPD扩增,其中对照组共扩增出65条带。与对照组相比,离子液体处理组的RAPD图谱变化表现在条带亮度增强或减弱以及新带的出现和正常带的消失。各处理组多态性带及变异带的总数随离子液体烃链碳原子数增加而增加,而基因组模板稳定性则随离子液体烃链长度增加而降低,这说明咪唑基离子液体对涡虫有遗传毒性,且随着离子液体烃链长度增加其对涡虫的遗传毒性增强。同时研究结果也证明RAPD分析是一种检测环境污染物对动物DNA损伤的灵敏方法。     

RAPD方法鉴定油菜“杂油59”种子纯度的研究

本文采用ＲＡＰＤ分子标记技术对我国育成的油菜新品种“杂油５９”的亲本和Ｆ１代种子纯度进行了技术鉴定。用４０个１０ｍｅｒ的随机引物对“杂油５９”的２个亲本“垦Ｃ８”和“陕３Ａ”进行了ＲＡＰＤ分析,共出现２９０条带,分布于３５３０～２２０ｂｐ之间,大部分条带在双亲之间是相同的。引物Ｏｐａ－Ｏ６,Ｏｐｃ－１２,Ｏｐｃ－２０,Ｏｐｋ－０３,ｏｐｋ－１３,Ｏｐｊ－１２出现差异扩增带,经实验确认Ｏｐｋ－０３的