Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

中国红树科7种红树植物遗传多样性分析

以红树、红海榄、秋茄、角果木、木榄、海莲、尖瓣海莲等7种红树科植物为材料,采用改进的CTAB法获得了纯度较高、得率高、片段完整的基因组DNA。通过筛选出的15个有效引物进行RAPD分析,探讨了7种树植物间的亲缘关系。15个有效引物共扩增出617条DNA带,其中多态性条带415条,占总扩增条带的67.26%。利用Nei指数法得出7个分类群间的遗传一致度和遗传距离,并运用UPGMA法进行聚类分析。7个分类群分为A、B两个大组,平均遗传距离为0.41。将得出的7个分类群的DNA分子分类系统图,与传统的分类进行比较,发现结果相符。同时获得一个OPG05－900的差异片段可作为区分海莲和尖瓣海莲的分子标记。     

用RAPD和AFLP的方法对中国卤虫(Artemia)种及亲缘关系的研究

利用ＲＡＰＤ（随机扩增多态ＤＮＡ）和ＡＦＬＰ（扩增片段长度多态性）技术对不同种及种群卤虫的关系进行分析。 １０１个随机引物对４种卤虫Ａｆｒａｎｃｉｓｃａｎａ、Ａ ｕｒｍｉａｎａ、Ａ ｓｉｎｉｃａ和Ａ．ｐａｒｔｈｅｎｏｇｅｎｅｌｉｃａ基因组ＤＮＡ进行扩增,平均每个种获得７５１条带,其中４５８条带为多态性标记,每个引物提供平均７４个标记信息,聚类结果表明Ａ．ｓｉｎｉｃａ是不同于其他旧大陆两性生殖卤虫的一个独立的种。对来自 １５个种及品系的卤虫的 ＡＦＬＰ分析显示了非常好的遗传多态性,采用 １２对引物检测到 ５９４条带,其中 ４８０个为多态性标记。聚类结果表明来自西藏的两性生殖卤虫为不同于中国内陆两性生殖卤虫的新种。而孤雌生殖卤虫在进化过程中可能是多源的,中国内陆和沿海的孤雌生殖卤虫是沿着不同的途径进化的,内陆和沿海的孤雌生殖卤虫可能为不同的种。     

Molecular phylogeny of mangroves V. Analysis of genome relationships in mangrove species using RAPD and RFLP markers

 DNA from pooled leaf samples of 11 true major mangrove, three true minor mangrove, two mangrove associate, two mangrove parasite, three terrestrial and one cultivated species were isolated for the present study. In total, 198 random amplified polymorphic DNAs (RAPDs) and 180 restriction fragment length polymorphism (RFLP) loci were scored by using ten primers and 14 enzyme-probe combinations respectively. The polymorphism observed for these markers revealed a high degree of genetic diversity in mangroves at both inter-specific or inter-generic levels. A dendrogram, constructed after pooling both RAPD and RFLP data, using a similarity index was analysed for genome relationships among these species. The dendrogram showed clustering of all the major mangroves, except for Nypa fruticans (Arecaceae), into one group. All species under the tribe Rhizophorae formed a sub-cluster, to which Xylocarpus granatum was found to be the most closesly related species. The clustering pattern implied that Excoecaria agallocha and Acanthus ilicifolius should be considered as true minor mangroves. The present study also provided molecular data favouring the separation of Avicennia spp. from the Verbenaceae to create a monotypic family the Avicenniaceae. The separation of Viscum orientale into the Viscaceae was also favoured. Received: 5 March 1998 / Accepted: 1 April 1998     

Genetic relationships among ten endod types as revealed by a combination of morphological,RAPD and AFLP markers

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

Molecular characterization of intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using DNA based molecular markers

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity of J. curcas germplasm. In the present study, efforts were made to analyze the genetic diversity among the elite germplasms of J. curcas, selected on the basis of their performance in field using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The plants were selected on the basis of height, canopy circumference, number of seeds per fruit, weight of 100 seeds, seed yield in grams per plant and oil content. Out of 250 RAPD (with 26 primers), 822 AFLP (with 17 primers) and 19 SSR band classes, 141, 346 and 7 were found to be polymorphic, respectively. The percentage polymorphism among the selected germplasms using RAPD, AFLP and SSR was found to be 56.43, 57.9, and 36.84, respectively. The Jaccard’s similarity coefficient was found 0.91, 0.90 and 0.91 through RAPD, AFLP and SSR marker systems, respectively. Principle component analysis (PCA) and dendrogarm analysis of genetic relationship among the germplasm using RAPD, AFLP and SSR data showed a good correlation for individual markers. The germplasm JCC-11, 12, 13, 14 and 15 whose yield found to be high were clustered together in dendrogram and PCA analysis though JCC11 is geographically distinct from others. In overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and JCC 6 and JCC10 (in SSR) were found genetically diverse. Characterization of geographically distinct and genetically diverse germplasms with varied yield characters is an important step in marker assisted selection (MAS) and it can be useful for breeding programs and QTL mapping.     

A Comparison of AFLP and RAPD Markers for Genetic Diversity and Cultivar Identification in Cotton

AFLP and RAPDmarkers were employed in sixteen diploid cotton (Gossypium sp) cultivars for genetic diversity estimation and cultivar identification. Polymorphism information content (PIC) and percent polymorphism were found to be more for AFLP markers as compared to RAPD markers. Average Jaccard’s genetic similarity index was found to be almost similar using either AFLP or RAPD markers. All the cultivars could be distinguished from one another using AFLP markers and also by the combined RAPD profiles. Cultivar identification indicators like resolving power, marker index and probability of chance identity of two cultivars suggested the usefulness of AFLP markers over the RAPD markers. AFLP and RAPD analyses revealed limited genetic diversity in the studied cultivars. Cluster analysis of both RAPD and AFLP data produced two clusters, one containing cultivars of G. herbaceum and another containing cultivars of G. arboreum species. Highly positive correlation between cophenetic matrices using RAPD and AFLP markers was observed. AFLP markers were found to be more efficient for genetic diversity estimation, polymorphism detection and cultivar identification.     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵（Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较。26个RAPD引物产生了总计192条DNA条带,大小分布 于0.26kb-1.98kb之间,其中165条（86．12％）具有多态性,每条引物产生DNA条带的平均数为7．38。8对AFLP引物组合共产生了576条带,分布于100bp-500bp之间,其中的341条具有多态性,多态百分率为76．00％,每对引物组合产生DNA条带的平均数为72。RAPD方法检测的每位点有效等位基因数（1．76）大于AFLP（1．65）,AFLP标记位点的平均多态性信息量（PIC）（0．38）低于RAPD标记位点PIC（0．41）,但AFLP标记具有很高的多态性检测效率（Ai=38．52）。用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47．84％－82．06％,平均相似性系数为0．6495,而采用AFLP的Nei氏相似性系数分布在54．15％－83．52％,平均相似性系数为0．6884。RAPD数据的标准差为0．13,而AFLP数据的标准差为0．08。因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反。源于两种不同标记的遗传相似矩阵的相关系数为0．51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性,无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群。     

A genetic map of Maritime pine based on AFLP, RAPD and protein markers

TheAFLP (amplified fragment length polymorphism) technique was adapted to carry out genetic analysis in maritime pine, a species characterized by a large genome size (24 pg/C). A genetic linkage map was constructed for one F₁ individual based on 239 AFLP and 127 RAPD (randomly amplified polymorphic DNA) markers. Markers were scored on megagametophytes (1n) from 200 germinated F₂ seedlings. Polymorphism rate, labour time and cost of both AFLP and RAPD techniques were compared. The AFLP technique was found to be twice as fast and three-times less costly per marker than the RAPD technique. Thirteen linkage groups were identified with a LOD score ≥6 covering 1873 cM, which provided 93.4% of genome coverage. Proteins were extracted from needles (2n) of the F₂ progeny and revealed by 2-DE (two-dimensional electrophoresis). Thirty one segregating proteins were mapped using a QTL detection strategy based on the quantification of protein accumulation. Two framework maps of the same F₁ individual are now available. The first map (Plomion et al. 1996) uses RAPD markers and the second map, presented in this study, uses mostly AFLP markers. Although the total genetic length of both maps was almost identical, differences among homologous groups were observed. Received: 11 February 1999 / Accepted: 29 April 1999     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

Genetic linkage maps of white birches (<Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk. and <Emphasis Type="Italic">B. pendula</Emphasis> Roth) based on RAPD and AFLP markers

A pseudo-testcross mapping strategy was used in combination with the random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) genotyping methods to develop two moderately dense genetic linkage maps for Betula platyphylla Suk. (Asian white birch) and B. pendula Roth (European white birch). Eighty F₁ progenies were screened with 291 RAPD markers and 451 AFLP markers. We selected 230 RAPD and 362 AFLP markers with 1:1 segregation and used them for constructing the parent-specific linkage maps. The resultant map for B. platyphylla was composed of 226 markers in 24 linkage groups (LGs), and spanned 2864.5 cM with an average of 14.3 cM between adjacent markers. The linkage map for B. pendula was composed of 226 markers in 23 LGs, covering 2489.7 cM. The average map distance between adjacent markers was 13.1 cM. Clustering of AFLP markers was observed on several LGs. The availability of these white birch linkage maps will contribute to the molecular genetics and the implementation of marker-assisted selection in these important forest species.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

Conservation genetics of endangeredBrasenia schreberi based on RAPD and AFLP markers

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θ^B) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (N_em), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoringB. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Identification and Characterization of Malaysian River Catfish, Mystus nemurus (C&V): RAPD and AFLP Analysis

This work represents the first application of the amplified fragment length polymorphism (AFLP) technique and the random amplified polymorphic DNA (RAPD) technique in the study of genetic variation within and among five geographical populations of M. nemurus. Four AFLP primer combinations and nine RAPD primers detected a total of 158 and 42 polymorphic markers, respectively. The results of AFLP and RAPD analysis provide similar conclusions as far as the population clustering analysis is concerned. The Sarawak population, which is located on Borneo Island, clustered by itself and was thus isolated from the rest of the populations located in Peninsular Malaysia. Both marker systems revealed high genetic variability within the Universiti Putra Malaysia (UPM) and Sarawak populations. Three subgroups each from the Kedah, Perak, and Sarawak populations were detected by AFLP but not by RAPD. Unique AFLP fingerprints were also observed in some unusual genotypes sampled in Sarawak. This indicates that AFLP may be a more efficient marker system than RAPD for identifying genotypes within populations.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

Analysis of genetic diversity in Phytophthora colocasiae causing leaf blight of taro (Colocasia esculenta) using AFLP and RAPD markers

The oomycetous fungus Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro and is widely distributed in India. Molecular and cultural techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae obtained from different geographical regions of India. Analysis of the 5.8-ITS region revealed detectable intraspecific variation among isolates. Ten random amplified polymorphic DNA (RAPD) and eight amplified fragment length polymorphism (AFLP) primers produced 198 and 510 reproducible fragments, respectively. AFLP produced 100 % polymorphism, whereas RAPD showed 93.5 % polymorphism. The average value of the number of observed alleles, the number of effective alleles, mean Nei’s genetic diversity, and Shannon’s information index were 2.00–1.94, 1.53–1.36, 0.31–0.24, and 0.47–0.40, respectively, for two DNA markers used. Analysis of molecular variance (AMOVA) for both markers produced similar results with the majority (85 %, AFLP; 89 %, RAPD) of the diversity present within population of P. colocasiae. Dendrograms based on two molecular data using the unweighted pair group method with arithmetic mean (UPGMA) was incongruent and classified the P. colocasiae isolates into one and two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r?=?0.904) and AFLP (r?=?0.825). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.