ATG-anchored AFLP (ATG-AFLP) analysis in cotton

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.     

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.     

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.     

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.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

AFLP标记技术在鉴定甘蓝种子真实性及品种纯度中的应用

利用AFLP方法对在农业生产上大面积推广使用的5个甘蓝杂交组合及其10个亲本共15个材料进行了分析研究,得到了清晰的DNA扩增指纹图谱.在AFLP分析中,共采用了32个EcoRI+3/MseI+3引物组合,每个引物组合扩增出的条带数在43～62条之间,平均为54.5条.并从这32个引物组合中筛选出一个引物组合,能够用来对供试5个甘蓝杂交种进行真实性及品种纯度鉴定.从而证明了AFLP技术在甘蓝种子真实性及品种纯度鉴定中的可行性.该技术应用于甘蓝种子真实性及品种纯度鉴定在国内尚属首次,其稳定性重复性好,检测分辨率高,很适合甘蓝种子真实性及品种纯度鉴定.是今后种子真实性及品种纯度鉴定的发展方向,应加大其研究力度.     

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.     

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.     

Saturation of two chromosome regions conferring resistance to SCMV with SSR and AFLP markers by targeted BSA

Quantitative trait loci (QTLs) and bulked segregant analyses (BSA) identified the major genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3, conferring resistance against sugarcane mosaic virus (SCMV) in maize. Both chromosome regions were further enriched for SSR and AFLP markers by targeted bulked segregant analysis (tBSA) in order to identify and map only markers closely linked to either Scmv1 or Scmv2. For identification of markers closely linked to the target genes, symptomless individuals of advanced backcross generations BC5 to BC9 were employed. All AFLP markers, identified by tBSA using 400 EcoRI/ MseI primer combinations, mapped within both targeted marker intervals. Fourteen SSR and six AFLP markers mapped to the Scmv1 region. Eleven SSR and 18 AFLP markers were located in the Scmv2 region. Whereas the linear order of SSR markers and the window size for the Scmv2 region fitted well with publicly available genetic maps, map distances and window size differed substantially for the Scmv1 region on chromosome 6. A possible explanation for the observed discrepancies is the presence of two closely linked resistance genes in the Scmv1 region.     

Genetic variation in Echinacea angustifolia along a climatic gradient

BACKGROUNDS AND AIMS: Echinacea angustifolia is a widespread species distributed throughout the Great Plains region of North America. Genetic differentiation among populations was investigated along a 1500 km north-south climatic gradient in North America, a region with no major geographical barriers. The objective of the study was to determine if genetic differentiation of populations could be explained by an isolation-by-distance model or by associations with climatic parameters known to affect plant growth and survival. METHODS: Historical climatic data were used to define the nature of the climatic gradient and AFLP markers were used to establish patterns of population genetic differentiation among ten Echinacea populations collected from North Dakota to Oklahoma. A total of 1290 fragments were scored using six EcoRI/MseI and three PstI/MseI primer combinations. Assessment of the correlation between climatic, genetic and geographic distances was assessed by Mantel and partial Mantel tests. KEY RESULTS: PstI/MseI combinations produced significantly fewer fragments, but a larger percentage was unique compared with EcoRI/MseI markers. Using estimates of F(ST), populations in Oklahoma and southern Kansas were identified as the most divergent from the other populations. Both the neighbour-joining tree and principal co-ordinate analysis clustered the populations in a north-south spatial orientation. About 60% of the genetic variation was found within populations, 20% among populations and the remaining 20% was partitioned among groups that were defined by the topology of the neighbour-joining tree. Significant support was found for the isolation-by-distance model independent of the effects of annual mean precipitation, but not from annual mean temperature and freeze-free days. CONCLUSIONS: Echinacea angustifolia populations exhibit genetic divergence along a north-south climatic gradient. The data support an isolation-by-distance restriction in gene flow that is independent of annual mean precipitation.     

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.     

Barley chromosome addition lines of wheat for screening of AFLP markers on barley chromosomes.

We conducted AFLP (Amplified Fragment Length Polymorphism) analysis with the six wheat-barley chromosome addition lines of common wheat cultivar Chinese Spring. We analyzed the AFLP fingerprints generated by 36 combinations of selective-amplification primers to find 103 markers specific to the barley chromosomes (2.9 markers per combination on average). The numbers of AFLP markers mapped to the barley chromosomes varied (one to 16) depending of the primer combinations. Each barley chromosome had 10 to 27 AFLP markers (17.2 markers on average). We identified the chromosome arms in which these markers are located using the barley telocentric addition lines (one to 20 markers per chromosome arm). The AFLP markers were not distributed evenly among chromosomes and chromosome arms. We could not determine the chromosome-arm locations for some of the barley-specific markers, either because such markers were found in both the short- and long-arm telocentric lines, or in neither line.     

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.     

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.     

Toward a marker-dense meiotic map of the potato genome: lessons from linkage group I

Segregation data were obtained for 1260 potato linkage group I-specific AFLP loci from a heterozygous diploid potato population. Analytical tools that identified potential typing errors and/or inconsistencies in the data and that assembled cosegregating markers into bins were applied. Bins contain multiple-marker data sets with an identical segregation pattern, which is defined as the bin signature. The bin signatures were used to construct a skeleton bin map that was based solely on observed recombination events. Markers that did not match any of the bin signatures exactly (and that were excluded from the calculation of the skeleton bin map) were placed on the map by maximum likelihood. The resulting maternal and paternal maps consisted of 95 and 101 bins, respectively. Markers derived from EcoRI/MseI, PstI/MseI, and SacI/MseI primer combinations showed different genetic distributions. Approximately three-fourths of the markers placed into a bin were considered to fit well on the basis of an estimated residual "error rate" of 0-3%. However, twice as many PstI-based markers fit badly, suggesting that parental PstI-site methylation patterns had changed in the population. Recombination frequencies were highly variable across the map. Inert, presumably centromeric, regions caused extensive marker clustering while recombination hotspots (or regions identical by descent) resulted in empty bins, despite the level of marker saturation.     

Detection of DNA polymorphism in Papaver somniferum genotypes differing in straw morphinan alkaloid content

Abstract

Thirty‐two distinct accessions of Papaver somniferum were screened for morphinan alkaloid content in the straw. The combined content of major morphinan alkaloids (morphine+codeine+thebaine) was found to vary in the range 0.2260–0.0683%. Two genotypes each, were selected as prototypes for low [I‐48 (0.0683%) and I‐344 (0.0878%)] and high [Pps‐1 (0.2260%) and N‐3 (0.2074%)] morphinan alkaloid content for studying DNA polymorphism. RAPD analysis of these four genotypes using 80 primers could not detect the polymorphism. However, AFLP analysis of these genotypes with 12 EcoRI/MseI primer pairs could distinctly group the high‐ and low‐morphinan alkaloid genotypes separately. Furthermore, 50 AFLP fragments, specific to high‐straw morphinan alkaloid genotypes (Pps‐1 and N‐3) and 28 DNA fragments specific to low‐straw morphinan alkaloid genotypes (I‐48 and I‐344) could be identified. This investigation is the first report on the polymorphism identified in the genotypes differing in their straw morphinan alkaloid content. This DNA polymorphism could be exploited for defining chemotypes at an early seedling stage in poppy breeding programmes.     

60份优质番茄自交系遗传多样性AFLP分析

本文以AFLP技术对60份优质番茄自交系的遗传多样性进行了分析。17对AFLP引物组合共扩增得到905条带,其中多态性条带251条,平均每对引物检测到约15个多态性位点,平均多态率为27.7%;仅用E7M4和E7M10两对引物组合就可以绘制60份番茄自交系的指纹图谱。60个自交系间的Jaccard遗传相似系数变幅为0.259-0.952,平均值为0.664。采用UPGMA方法聚类,遗传相似系数变幅为0.54-0.57时,60份番茄材料可以划分为7个类群。聚类结果与来源、果实大小、果形等性状未表现出明显的相关性。     

AFLP Analysis Confirms Exclusive Maternal Genomic Contribution of Meiogynogenetic Sea Bass (Dicentrarchus labrax L.)

Meiogynogenesis was induced in the European sea bass Dicentrarchus labrax L. by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division by a cold shock. Putative gynogenetic progeny derived from three groups of breeders were analyzed for maternal inheritance using amplified fragment length polymorphism (AFLP) markers. Discrimination of fingerprints was based on male-specific bands, which were absent in females. Four of 64 MseI/EcoRI primer pairs used to analyze parental polymerase chain reaction products were selected to screen progeny for paternal AFLP markers in each group. Four to 11 diagnostic bands per fish confirmed the gynogenetic origin of the progeny. AFLP analysis determined that 89.5%, 100%, and 100% of the sea bass from groups 1, 2, and 3, respectively, were gynogenetic. Our results show that AFLP analysis is suitable for verification of gynogenesis in fish.