Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton

In certain plant species including cotton (Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes (G. hirsutum) and three Pima cotton (G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting.     

Customisation of AFLP analysis for cassava varietal identification.

Amplified fragment length polymorphism (AFLP) markers were used in the characterization of eight cassava varieties. This nonradioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 3' end of primers. EcoRI/MseI and HindIII/MseI fragments generally gave monomorphic profiles while ApaI/TaqI fragments produced polymorphic profiles suggesting a genome with high G + C content. It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer. For cassava, the AFLP system provided a higher number of loci detected per run when compared to RAPD. The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties.     

Chromosomal location of AFLP markers in common wheat utilizing nulli-tetrasomic stocks.

Amplified fragment length polymorphism (AFLP) markers with a total of 256 EcoRI + ANN - MseI + CNN primer combinations were investigated employing the common wheat cultivar Triticum aestivum 'Chinese Spring.' On average, 103 fragments per primer combination were amplified, ranging from a maximum of 226 fragments to a minimum of 18 fragments. The primer combinations E + AAA - M + CNN and E + ATT - M + CNN produced very few distinct fragments. By using 15 randomly chosen EcoRI + ANN - MseI + CNN primer combinations, 928 AFLP markers were allocated to wheat chromosomes, of which 131 were assigned to specific chromosome arms. These AFLP markers were locus-specific and randomly distributed on the different chromosomes. In addition, 6 and 41 AFLP markers were simultaneously absent in two nulli-tetrasomics (NTs) of both homoeologous and non-homoeologous groups, respectively, whereas additional fragments were detected in N1BT1A, N5AT5D, and N6BT6A lines.     

A high-density linkage map in <Emphasis Type="Italic">Brassica juncea</Emphasis> (Indian mustard) using AFLP and RFLP markers

A high-density genetic linkage map of Brassica juncea (2n = 36) was constructed with 996 AFLP (amplified fragment length polymorphism) and 33 RFLP (restriction fragment length polymorphism) markers using a F1-derived doubled-haploid (DH) population of 123 individuals. This mapping population was developed by crossing a well-adapted, extensively grown Indian variety Varuna and a canola quality line Heera. The two lines are highly divergent and contain a number of contrasting qualitative and quantitative traits of high agronomic value. AFLPs were generated by the use of restriction enzymes EcoRI or PstI in combination with either MseI or TaqI. Using 91 primer pairs, a total of 1,576 parental polymorphic bands were detected of which 996 were used for mapping. In addition, 33 RFLP markers, developed from genomic clones of B. napus, were added to the map. The segregation of each marker and linkage analysis was performed using the program JoinMap version 2.0. The 1,029 mapped-markers were aligned in 18 linkage groups, which is the haploid chromosome number of the species, at LOD values ranging from 5 to 8. The total map length was 1,629 cM with an average marker interval of 3.5 cM. AFLP markers generated by EcoRI were more clustered, whereas PstI markers showed more extensive distribution. A set of 26 primer pairs (9 EcoRI/ MseI, 6 EcoRI/ TaqI, 6 PstI/ MseI and 5 PstI/ TaqI) generating 385 markers were identified for AFLP-based whole-genome selection as these markers covered 96% of the genome mapped with the 91 primer pairs. The map developed in the present study could be used for dissection and the transfer of agronomically important traits and favourable QTLs from ill-adapted exotic germplasm to cultivated Indian varieties.     

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.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

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.     

AFLP™ markers for DNA fingerprinting in cattle

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with Eco RI and Mse I is suggested, in mammals the enzyme combination Eco RI/ Taq I produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 Eco RI/ Taq I primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0·31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.     

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.     

Identification of Molecular Markers Associated with Semigamy in Cotton

Semigamy, controlled by one incomplete dominant gene (Se) in Pima cotton (Gossypium barbadense), is an aberrant form of sexual reproduction or apomixis, in which the sperm and egg nuclei fail to fuse and proceed to develop independently resulting in various outcomes, such as haploids, diploids, and chimeras. The objective of this study was to develop molecular markers linked to the semigamy gene using a (Pima S-1?×?57-4)?×?Pima S-1 BC₁F₁ mapping population consisting of 34 semigametic and 30 non-semigametic progenies. Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), ATG-anchored AFLP (ATG-AFLP), sequenced tagged site (STS), and single-strand conformational polymorphism (SSCP) markers were tested. None of the apparent polymorphic RAPD, AFLP, and ATG-AFLP markers were linked to the semigamy gene, while three polymorphic markers developed from genes based on comparative microarray and differential display analyses, SSCP Se18-1800 and Se18-3000, and STS Se19, were mapped to a linkage group carrying the semigamy gene. The results present evidence that targeted gene mapping is an efficient method using markers developed from comparative gene expression studies. The current work represents the first study in identifying DNA markers associated with semigamy in plants, providing an incentive toward high-resolution mapping and isolation of the semigamy gene.     

棉花2个多标记基因系及其杂交后代AFLP分析

应用AFLP分子标记技术,对陆地棉两个多标记基因系T582和T586及其杂交后代F1等进行了DNA多态性分析。结果表明:在58对EcoRI/MseI引物组合中,筛选出41对引物组合具有多态性,多态性的引物组合占筛选总组合的70.69%。AFLP分子标记具有高度的多态性,非常适于基因组差异较小的(棉花)材料之间的多态性筛选。采用聚丙烯酰胺银染法显带技术,AFLP进行PCR扩增能看到30~80条DNA亮带,且检测灵敏度高,可区别只相差十几个bp甚至几个bp大小的DNA片段。但AFLP标记以显性标记占绝对优势,共显性标记比率极少,故而难以区分种质的杂合和纯合,这是它的惟一不足之处。     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Hybridization in the section Mentha (Lamiaceae) inferred from AFLP markers

The amplified fragment length polymorphism (AFLP) method was used to evaluate genetic diversity and to assess genetic relationships within the section Mentha in order to clarify the taxonomy of several interspecific mint hybrids with molecular markers. To this end, genetic diversity of 62 Mentha accessions from different geographic origins, representing five species and three hybrids, was assessed. Three EcoRI/MseI AFLP primer combinations generated an average of 40 AFLP markers per primer combination, ranging in size from 50 to 500 base pairs (bp). The percentage of markers polymorphic ranged from 50% to 60% across all accessions studied. According to phenetic and cladistic analysis, the 62 mint accessions were grouped into two major clusters. Principal coordinates analysis separated species into well-defined groups, and clear relationships between species and hybrids could be described. Our AFLP analysis supports taxonomic classification established among Mentha species by conventional (morphological, cytological, and chemical) methods. It allows the assessment of phenetic relationships between species and the hybrids M. spicata and M. × piperita, largely cultivated all over the world for their menthol source, and provides new insights into the subdivision of M. spicata, based for the first time on molecular markers.     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Identification of AFLP and microsatellite markers linked with an aluminium tolerance gene in barley ( Hordeum vulgare L.)

Barley is the most sensitive among the cereals to aluminium (Al) stress and breeding for more tolerant cultivars is a priority. To enhance selection efficiency for Al tolerance in barley, PCR-based AFLP and microsatellite markers linked to a locus conferring tolerance to aluminium were identified. The study used F(2) progeny derived from a single cross between Yambla (moderately tolerant of Al) and WB229 (tolerant of Al) and developed hydroponic pulse-recovery screening methods to assess tolerance of phenotypes based on root growth. The segregation ratios of tolerant and sensitive genotypes and F(3) progeny testing suggest that a single major gene controlled Al tolerance ( Alt). In order to determine the chromosomal location of the Alt gene, we used the AFLP technique coupled with bulk segregant analysis. We evaluated tolerant and sensitive bulks using 30 combinations of EcoRI/ MseI primers, and 12 of these permitted differentiation of the sensitive and tolerant bulks. More than 1,000 amplified fragments were obtained, and 98 polymorphic bands were scored. AFLP analysis of wheat-barley chromosome addition lines indicated that the Alt gene was located on barley chromosome 4H. Four chromosome 4H-specific microsatellite markers (Bmac310, Bmag353, HVM68 and HVMCABG) were tightly linked to Alt. The large allelic variation detected with microsatellite marker Bmag353 allowed us to implement this marker for routine marker-assisted selection for Al tolerance, and 396 plants could be screened on a single gel.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

基于AFLP分子标记的桂花品种核心种质的构建

用100个桂花品种荧光AFLP分子标记信息构建核心种质。利用获得的指纹信息,运用UPGMA聚类取样法,采用Kimura 2-parameter遗传距离,多次聚类随机抽样。结果表明:(1)8对引物共获得514条带,平均每对引物获得64条带。(2)从100个桂花品种中筛选了30个样本的核心种质。(3)比较核心种质和全部种质的Shan-non-Wiener指数(H′)和Simpson指数(D),t检验值均说明核心种质的遗传多样性指数与全部种质遗传多样性没有明显差异,表明所构建的核心样品能够很好地保留原始100个桂花品种的遗传多样性。     

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。