Phytochemical variation within populations of Echinacea angustifolia (Asteraceae)

Quantitative evaluation of phytochemical diversity in Echinacea angustifolia DC. populations from different natural geographic areas supports the existence of distinct natural chemotypes within the species. Consumers, growers and manufacturers of phytomedicines are interested in chemotype identification for prediction of phytochemical content in cultivar development. Six month old E. angustifolia roots, grown from nine different wild seed sources in a controlled environment, were extracted into 70% ethanol and 28 reported phytochemicals were measured by HPLC separation. Two-way ANOVA between the nine populations revealed quantitative differences (p<0.05) in the caffeic acid derivatives 2,3-O-dicaffeoyl tartaric acid (cichoric acid), 2-O-caffeoyl tartaric acid (caftaric acid), 1,3-dicaffeoyl-quinic acid (cynarin), echinacoside and ten reported alkamides. Canonical discriminant analysis determined the phytochemical variables which contributed the most towards chemotype distinction for five of the nine populations: undeca-2E,4Z-diene-8,10-diynoic acid-2-methylbutylamide¹, dodeca-2E,4E-dienoic acid isobutylamide¹, dodeca-2E-ene-8,10-diynoic acid isobutylamide⁷, hexadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide¹, cichoric acid⁷, caftaric acid¹, and echinacoside⁷ (¹p<0.0001, ⁷p<0.05). Five of those compounds were also significantly associated with latitudinal variation by regression analyses (p<0.05).     

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.     

Effect of temperature on stability of marker constituents in Echinacea purpurea root formulations.

Stability of an alkamide and a phenolic phytochemical marker in a hydro-alcoholic extract of Echinacea purpurea root and a dried powder prepared by evaporation of the extract was assessed in storage for 7 months at three temperature regimes: -20, 25 and 40 degrees Celsius. In the extract, the major alkamide, dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutyl amide, was not significantly affected by storage at any of the temperatures, but cichoric acid content declined as significantly (P = 0.05) at both 25 degrees C and 40 degrees C as compared to low-temperature storage. In the powder, the major alkamide showed a significantly reduced level at 25 degrees C and 40 degrees C while cichoric acid did not decline significantly. These results suggest that more attention should be given to the effect of formulation and temperature on storage of Echinacea products.     

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.     

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

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。     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Intraspecies Genomic Groups in Enterococcus faecium and Their Correlation with Origin and Pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Genetic variation in Danish and Norwegian germplasm collections of hops

The germplasm collections of hops (Humulus lupulus L.) in Denmark and Norway are maintained in clonal archives funded by the national authorities. The plants have been collected over the last decades as part of a strategy to conserve plant genetic resources for future generations. The major part of the various collections consist of plants collected in villages and gardens. About 20% are plants used for breeding, mainly kept in a collection at Carlsberg, Denmark. In order to identify any duplicates and with a view to learning more about the various collections, a DNA fingerprinting study was initiated, analysing 62 Danish and 34 Norwegian clones with a set of five amplified fragment length polymorphism (AFLP) markers. The AFLP analyses resulted in 41 polymorphic bands and were able to separate the majority of the Danish and the Norwegian accessions. UPGMA dendrograms showed 21 accession groups, and potential duplicates were found within 13 of these groups. Principal coordinates analysis revealed that plants were differentiated according to country of origin. In addition, regional separation of the plants within each country was also detected, and similar levels of diversity were found in the Danish and the Norwegian collections. Compared to the rest of the plants, there was less diversity within the Carlsberg material. For the Norwegian as well as a part of the Danish collection, morphological characterisation and chemical analysis was carried out, allowing a comparison of these to the AFLP data. A correlation with AFLP bands and both morphological and chemical characteristics was detected. The most promising results for further breeding was an association of AFLP bands with the content of colupulon in the cones, measured by relative values compared to the total alpha acid content. Further studies are needed to verify such an association with the potential to develop a PCR-based marker for hop breeding carried out in the clones now analysed with AFLP markers, making it possible to search for any association between AFLP data and phenotypic data.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Morphological and phytochemical variation, genetic structure and phenology in an introgressive hybrid swarm of Senecio hercynicus and S. ovatus (Compositae, Senecioneae)

Introgressive hybridisation between Senecio hercynicus and Senecio ovatus (Compositae, Senecioneae) was studied in a mixed stand of the two species on the S- and SW-facing slopes of the German part of the Bohemian Forest (SE Germany). Morphological variation based on multivariate analysis of 14 diagnostic characters, along with genetic data from an amplified fragment length polymorphism (AFLP) fingerprinting analysis, indicates that the surveyed stand forms an intensively introgressed hybrid swarm. The majority of individuals were found to be intermediate between the two parental taxa, and strong statistical correlation between phenetic and genetic distances was observed. In contrast to that, flowering time of individuals (expressed as the time when 50% of the capitula of a plant were in flower) was found to follow a bimodal distribution in the hybrid swarm and lacked any correlation with the genetic and morphological relationships among plants. The same was true for the spectra of pyrrolizidine alkaloids (PA) revealed by a gas-chromatographic survey: most of the 142 individuals surveyed fell into one of two main chemotypes, only a few plants exhibited an intermediate and additive PA spectrum, and no correlation with the genetic and morphological relationships among plants was observed. Assuming that most of the AFLP markers are neutral, we conclude that the correlation of morphology with the genetic pattern may argue for the neutrality of morphological features analysed. Consequently, we interpret the lack of correlation between phenological and phytochemical data with the neutral genetic markers as evidence that both phenology and phytochemistry may be either under selection or that these features are genetically or biosynthetically constrained.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

Genetic diversity analysis in sorghum germplasm as estimated by AFLP, SSR and morpho-agronomical markers

A comparison of the different methods of the estimation of genetic diversity is important to evaluate their utility as a tool in germplasm conservation and plant breeding. Amplified fragment length polymorphism (AFLP), microsatellites or SSR and morphological traits markers were used to evaluate 45 sorghum germplasm for genetic diversity assessment and discrimination power. The mean polymorphism information content (PIC) values were 0.65 (AFLPs) and 0.46 (SSRs). The average pairwise genetic distance estimates were 0.57 (morphological traits), 0.62 (AFLPs) and 0.60 (SSRs) markers data sets. The Shannon diversity index was higher for morphological traits (0.678) than AFLP (0.487) and SSR (0.539). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for AFLP and SSR markers, as well as for morphological and SSR markers were significantly related (p <0.001). However, morphological and AFLP data showed non-significant correlation (p >0.05). Both data sets from AFLP and SSR allowed all accessions to be uniquely identified; two accessions could not be distinguished by the morphological data. In summary, AFLP and SSR markers proved to be efficient tools in assessing the genetic variability among sorghum genotypes. The patterns of variation appeared to be consistent for the three marker systems, and they can be used for designing breeding programmes, conservation of germplasm and management of sorghum genetic resources.     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Evaluation of AFLP in Beta

 AFLP markers were evaluated for their usefulness in the genetic analysis of sugarbeet and wild Beta species. Accessions of ten different sugarbeet breeding lines and five wild beets were screened using 256 primer combinations. Of the 11 309 bands investigated, 96.4% were polymorphic among the accessions. A strong positive correlation was found between the number of polymorphisms and AT content of the selective bases of the primer combinations. Random subsets of primer combinations were used to produce genetic distance trees. Permutation tests showed that, for the wild beets, 500 AFLP bands sufficed to obtain the best topology of the tree with a probability at any given node of more than 99%. Ten times as many bands were necessary to obtain support values of the same order of magnitude for the sugarbeet lines. The reproducibility of AFLP for seven primer combinations was investigated by repeated analysis of all steps from DNA isolation to data scoring. For 5088 comparisons, the overall reproducibility was 97.6%. Robustness to genotyping errors was investigated by including an artificial F₁ (1 : 1 DNA mixture) of two sugarbeet lines in the screen for polymorphisms. For the 3160 cases of polymorphism between the two lines, 0.2% genotyping errors were found. The general reliability and usefulness of AFLP markers are discussed in relation to the results obtained. Received: 18 May 1998 / Accepted: 28 October 1998     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

AFLP在橡胶树优异种质研究中的应用

采用377DNA测序仪（P.E.Corp.)用AFLP技术对25种分别具有高产／低产、抗白粉病／感白粉病、抗寒／不抗寒、死皮／不死皮性状的橡胶树（Hevea brasiliensis Mull.Ary)无性系（其中Wickham种质15种,Amazon野生橡胶树种质10种）进行了指纹图谱分析,从64对引物组合中选出2对引物组合构建了所有被研究种质的指纹图谱。2对引物共扩增出518条带,多态性比率超过98．6％。遗传多样性分析表明,橡胶树种质间遗传距离为0．25－0．81,RRIM600种质内遗传距离为0．07－0．17。通过基因分型分析获得一条大小为320bp的抗白粉病种质特有的片段。根据以上的AFLP数据进行了聚类分析,结果表明所有被研究种质几乎是依次聚成一个大类。     

Primary Study of Rice AFLP Analysis-Optimization of Reaction Conditions and Analysis of Thermo sensitive Genic Male Sterile Rice Allelic Mutant Lines

AFLP analysis was performed between a pair of thermo-sensitive genic male sterile (TGMS) rice allelic mutant lines (5460S and 5460F). The reaction conditions for rice AFLP assay were optimized. The relative efficiencies for polymorphism detection of RFLP, RAPD and AFLP were compared. The results indicated that the efficiency for polymorphism detection in rice was in the order of AFLP > RAPD > RFLP, and also indicated that AFLP was a powerful DNA molecular marker technique for polymorphism detection, especially in the case of extremely low polymorphism, such as isogenic lines and allehc mutant hnes. Some of the AFLP products between the TGMS rice allehc mutant lines were cloned. Three of them were used as mixed probes to screen BAC library of rice line 5460S. 12 positive clones were screened out. In addition, the advantages and disadvantages of these three molecular marker systems were discussed.