Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat

In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC ( Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 x Opata 85 (ITMI pop). This population was used earlier for the construction of RFLP ( Restriction Fragment Length Polymorphism) maps in bread wheat (ITMI map). Using the ITMI pop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.     

Isolation and mapping of microsatellite markers specific for the D genome of bread wheat.

The potential of Aegilops tauschii, the diploid progenitor of the D genome of wheat, as a source of microsatellite markers for hexaploid bread wheat was investigated. By screening lambda phage and plasmid libraries of Ae. tauschii genomic DNA, dinucleotide microsatellites containing GA and GT motifs were isolated and a total of 65 functional microsatellite markers were developed. All primer pairs that were functional in Ae. tauschii amplified well in hexaploid wheat. Fifty-five loci amplified by 48 primer sets were placed onto a genetic framework map of the reference population of the International Triticeae Mapping Initiative (ITMI) 'Opata 85' x 'W7984'. The majority of microsatellite markers could be assigned to the chromosomes of the D genome of wheat. The distribution of the markers along the chromosomes is random. Chromosomal location of 22 loci nonpolymorphic in the reference population was determined using nullitetrasomic lines of Triticum aestivum 'Chinese Spring'. The results of this study demonstrate the value of microsatellite markers isolated from Ae. tauschii for the study of bread wheat. The microsatellite markers developed improve the existing wheat microsatellite map and can be used in a wide range of genetic studies and breeding programs.     

A high-density intervarietal map of the wheat genome enriched with markers derived from expressed sequence tags

Bread wheat (Triticum aestivum L.) is a hexaploid species with a large and complex genome. A reference genetic marker map, namely the International Triticeae Mapping Initiative (ITMI) map, has been constructed with the recombinant inbred line population derived from a cross involving a synthetic line. But it is not sufficient for a full understanding of the wheat genome under artificial selection without comparing it with intervarietal maps. Using an intervarietal mapping population derived by crossing Nanda2419 and Wangshuibai, we constructed a high-density genetic map of wheat. The total map length was 4,223.1 cM, comprising 887 loci, 345 of which were detected by markers derived from expressed sequence tags (ESTs). Two-thirds of the high marker density blocks were present in interstitial and telomeric regions. The map covered, mostly with the EST-derived markers, approximately 158 cM of telomeric regions absent in the ITMI map. The regions of low marker density were largely conserved among cultivars and between homoeologous subgenomes. The loci showing skewed segregation displayed a clustered distribution along chromosomes and some of the segregation distortion regions (SDR) are conserved in different mapping populations. This map enriched with EST-derived markers is important for structure and function analysis of wheat genome as well as in wheat gene mapping, cloning, and breeding programs.     

Genetic and physical mapping of new EST-derived SSRs on the A and B genome chromosomes of wheat

The availability of genetic maps and phenotypic data of segregating populations allows to localize and map agronomically important genes, and to identify closely associated molecular markers to be used in marker-assisted selection and positional cloning. The objective of the present work was to develop a durum wheat intervarietal genetic and physical map based on genomic microsatellite or genomic simple sequence repeats (gSSR) markers and expressed sequence tag (EST)-derived microsatellite (EST-SSR) markers. A set of 122 new EST-SSR loci amplified by 100 primer pairs was genetically mapped on the wheat A and B genome chromosomes. The whole map also comprises 149 gSSR markers amplified by 120 primer pairs used as anchor chromosome loci, two morphological markers (Black colour, Bla1, and spike glaucousness, Ws) and two seed storage protein loci (Gli-A2 and Gli-B2). The majority of SSR markers tested (182) was chromosome-specific. Out of 275 loci 241 loci assembled in 25 linkage groups assigned to the chromosomes of the A and B genome and 34 remained unlinked. A higher percentage of markers (54.4%), localized on the B genome chromosomes, in comparison to 45.6% distributed on the A genome. The whole map covered 1,605 cM. The B genome accounted for 852.2 cM of genetic distance; the A genome basic map spanned 753.1 cM with a minimum length of 46.6 cM for chromosome 5A and a maximum of 156.2 cM for chromosome 3A and an average value of 114.5 cM. The primer sets that amplified two or more loci mapped to homoeologous as well as to non-homoeologous sites. Out of 241 genetically mapped loci 213 (88.4%) were physically mapped by using the nulli-tetrasomic, ditelosomic and a stock of 58 deletion lines dividing the A and B genome chromosomes in 94 bins. No discrepancies concerning marker order were observed but the cytogenetic maps revealed in some cases small genetic distance covered large physical regions. Putative function for mapped SSRs were assigned by searching against GenBank nonredundant database using TBLASTX algorithms.     

A PCR-Based Genetic Map for Human Chromosome 3

Oligonucleotide primers for 125 simple sequence repeat microsatellite-based genetic markers have been assayed by polymerase chain reaction (PCR) in the CEPH reference family panel. These microsatellites include 101 dinucleotide repeats as well as 24 new tetranucleotide repeats. The average heterozygosity of this marker set was 72.4%. Genetic data were analyzed with the genetic mapping package LINKAGE. A subset of these microsatellite markers define a set of 56 uniquely ordered loci (>1000:1 against local inversion) that span 271 cM. Sixty-seven additional loci were tightly linked to markers on the uniquely ordered map, but could not be ordered with such high precision. These markers were positioned by CMAP into confidence intervals. One hundred thirteen of the microsatellite markers were also tested on a chromosome 3 framework somatic cell hybrid panel that divides this chromosome into 23 cytogenetically defined regions, integrating the genetic and physical maps of this chromosome. The high density, high heterozygosity, and PCR format of this genetically and physically mapped set of markers will accelerate the mapping and positional cloning of new chromosome 3 genes.     

An integrated SSR map of grapevine based on five mapping populations

A grapevine (mainly Vitis vinifera L., 2n = 38) composite genetic map was constructed with CarthaGene using segregation data from five full-sib populations of 46, 95, 114, 139 and 153 individuals, to determine the relative position of a large set of molecular markers. This consensus map comprised 515 loci (502 SSRs and 13 other type PCR-based markers), amplified using 439 primer pairs (426 SSRs and 13 others) with 50.1% common markers shared by at least two crosses. Out of all loci, 257, 85, 74, 69 and 30 were mapped in 1, 2, 3, 4 and 5 individual mapping populations, respectively. Marker order was generally well conserved between maps of individual populations, with only a few significant differences in the recombination rate of marker pairs between two or more populations. The total length of the integrated map was 1,647 cM Kosambi covering 19 linkage groups, with a mean distance between neighbour loci of 3.3 cM. A framework-integrated map was also built, with marker order supported by a LOD of 2.0. It included 257 loci spanning 1,485 cM Kosambi with a mean inter-locus distance of 6.2 cM over 19 linkage groups. These integrated maps are the most comprehensive SSR-based maps available so far in grapevine and will serve either for choosing markers evenly scattered over the whole genome or for selecting markers that cover particular regions of interest. The framework map is also a useful starting point for the integration of the V. vinifera physical and genetic maps.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

An integrated DArT-SSR linkage map of durum wheat

Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.     

Wide coverage of the tetraploid cotton genome using newly developed microsatellite markers

Microsatellite [simple-sequence repeat (SSR)] markers were developed and positioned on the genetic map of tetraploid cotton. Three hundred and ninety-two unique microsatellite sequences, all but two containing a (CA/GT) repeat, were isolated, and the deduced primers were used to screen for polymorphism between the Gossypium hirsutum and G. barbadense parents of the mapping population analyzed in our laboratory. The observed rate of polymorphism was 56%. The 204 polymorphic SSRs revealed 261 segregating bands, which ultimately gave rise to 233 mapped loci. The updated status of our genetic map is now of 1,160 loci and 5,519 cM, with an average distance between two loci of 4.8 cM. The presence of a total of 466 microsatellite loci, with an average distance of 12 cM between two SSR loci, now provides wide coverage of the genome of tetraploid cotton and thus represents a powerful means for the production of a consensus map and for the effective tracking of QTLs.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00122-004-1612-1Communicated by C. Möllers     

High-density genetic and physical bin mapping of wheat chromosome 1D reveals that the powdery mildew resistance gene <Emphasis Type="Italic">Pm24</Emphasis> is located in a highly recombinogenic region

Genetic maps of wheat chromosome 1D consisting of 57 microsatellite marker loci were constructed using Chinese Spring (CS) × Chiyacao F₂ and the International Triticeae Mapping Initiative (ITMI) recombinant inbred lines (RILs) mapping populations. Marker order was consistent, but genetic distances of neighboring markers were different in two populations. Physical bin map of 57 microsatellite marker loci was generated by means of 10 CS 1D deletion lines. The physical bin mapping indicated that microsatellite marker loci were not randomly distributed on chromosome 1D. Nineteen of the 24 (79.2%) microsatellite markers were mapped in the distal 30% genomic region of 1DS, whereas 25 of the 33 (75.8%) markers were assigned to the distal 59% region of 1DL. The powdery mildew resistance gene Pm24, originating from the Chinese wheat landrace Chiyacao, was previously mapped in the vicinity of the centromere on the short arm of chromosome 1D. A high density genetic map of chromosome 1D was constructed, consisting of 36 markers and Pm24, with a total map length of 292.7 cM. Twelve marker loci were found to be closely linked to Pm24. Pm24 was flanked by Xgwm789 (Xgwm603) and Xbarc229 with genetic distances of 2.4 and 3.6 cM, respectively, whereas a microsatellite marker Xgwm1291 co-segregated with Pm24. The microsatellite marker Xgwm1291 was assigned to the bin 1DS5-0.70-1.00 of the chromosome arm 1DS. It could be concluded that Pm24 is located in the ‘1S0.8 gene-rich region’, a highly recombinogenic region of wheat. The results presented here would provide a start point for the map-based cloning of Pm24.     

Development of a genome-wide anchored microsatellite map for common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A total of 150 microsatellite markers developed for common bean (Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican × Andean population, DOR364 × G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.Communicated by H.F. Linskens     

Linkage Map of the Honey Bee, Apis Mellifera, Based on Rapd Markers

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be ~3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.     

Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558

The present study describes a new set of 61 polymorphic microsatellite markers for beans and the construction of a genetic map using the BAT93 x Jalo EEP558 (BJ) population for the purpose of developing a reference linkage map for common bean (Phaseolus vulgaris). The main objectives were to integrate new microsatellites on the existing framework map of the BJ population, and to develop the first linkage map for the BJ population based exclusively on microsatellites. Of the total of 264 microsatellites evaluated for polymorphism, 42.8% showed polymorphism between the genitors. An integrated map was created totaling 199 mapped markers in 13 linkage groups, with an observed length of 1358 cM and a mean distance between markers of 7.23 cM. For the map constructed exclusively with microsatellites, 106 markers were placed in 12 groups with a total length of 606.8 cM and average distance of 6.8 cM. Linkage group designation and marker order for BM microsatellites generally agreed with previous mapping, while the new microsatellites were well distributed across the genome, corroborating the utility of the BJ population for a reference map. The extensive use of the microsatellites and the availability of a reference map can help in the development of other genetic maps for common bean through the transfer of information of marker order and linkage, which will allow comparative analysis and map integration, especially for future quantitative trait loci and association mapping studies.     

A high-density microsatellite consensus map for bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A microsatellite consensus map was constructed by joining four independent genetic maps of bread wheat. Three of the maps were F₁-derived, doubled-haploid line populations and the fourth population was Synthetic × Opata, an F₆-derived, recombinant-inbred line population. Microsatellite markers from different research groups including the Wheat Microsatellite Consortium, GWM, GDM, CFA, CFD, and BARC were used in the mapping. A sufficient number of common loci between genetic maps, ranging from 52 to 232 loci, were mapped on different populations to facilitate joining the maps. Four genetic maps were developed using MapMaker V3.0 and JoinMap V3.0. The software CMap, a comparative map viewer, was used to align the four maps and identify potential errors based on consensus. JoinMap V3.0 was used to calculate marker order and recombination distances based on the consensus of the four maps. A total of 1,235 microsatellite loci were mapped, covering 2,569 cM, giving an average interval distance of 2.2 cM. This consensus map represents the highest-density public microsatellite map of wheat and is accompanied by an allele database showing the parent allele sizes for every marker mapped. This enables users to predict allele sizes in new breeding populations and develop molecular breeding and genomics strategies.Electronic Supplementary Material Supplementary material is available for this article at     

Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Because of polyploidy and large genome size, deletion stocks of bread wheat are an ideal material for physically allocating ESTs and genes to small chromosomal regions for targeted mapping. To enhance the utility of deletion stocks for chromosome bin mapping, we characterized a set of 84 deletion lines covering the 21 chromosomes of wheat using 725 microsatellites. We localized these microsatellite loci to 94 breakpoints in a homozygous state (88 distal deletions, 6 interstitial), and 5 in a heterozygous state representing 159 deletion bins. Chromosomes from homoeologous groups 2 and 5 were the best covered (126 and 125 microsatellites, respectively) while the coverage for group 4 was lower (80 microsatellites). We assigned at least one microsatellite in up to 92% of the bins (mean 4.97 SSR/bin). Only a few discrepancies concerning marker order were observed. The cytogenetic maps revealed small genetic distances over large physical regions around the centromeres and large genetic to physical map ratios close to the telomeres. As SSRs are the markers of choice for many genetic and breeding studies, the mapped microsatellite loci will be useful not only for deletion stock verifications but also for allocating associated QTLs to deletion bins where numerous ESTs that could be potential candidate genes are currently assigned.     

Development and genetic mapping of sequence-tagged microsatellites (STMs) in bread wheat (Triticum aestivum L.)

The density of SSRs on the published genetic map of bread wheat (Triticum aestivum L.) has steadily increased over the last few years. This has improved the efficiency of marker-assisted breeding and certain types of genetic research by providing more choice in the quality of SSRs and a greater chance of finding polymorphic markers in any cross for a chromosomal region of interest. Increased SSR density on the published wheat genetic map will further enhance breeding and research efforts. Here, sequence-tagged microsatellite profiling (STMP) is demonstrated as a rapid technique for the economical development of anonymous genomic SSRs to increase marker density on the wheat genetic map. A total of 684 polymorphic sequence-tagged microsatellites (STMs) were developed, and 380 were genetically mapped in three mapping populations, with 296 being mapped in the International Triticeae Mapping Initiative W7984 × Opata85 recombinant inbred cross. Across the three populations, a total of 479 STM loci were mapped. Several technological advantages of STMs over conventional SSRs were also observed. These include reduced marker deployment costs for fluorescent-based SSR analysis, and increased genotyping throughput by more efficient electrophoretic separation of STMs and a high amenability to multiplex PCR.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends

Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala ‘Maxxa’, 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

花椰菜遗传图谱的构建及NBS-LRR类抗性同源基因在图谱中的定位

利用AFLP和NBS profiling技术, 以花椰菜自交系“AD白花”与高代自交不亲和系“C-8”杂交得到的F1代自交产生的F2代分离群体为材料, 构建了第一个花椰菜遗传连锁图谱。该图谱由234个AFLP标记和21个NBS标记构成了9个连锁群, 总图距为668.4 cM, 标记间平均距离为2.9 cM。每个连锁群包含的位点数从12到47个, 相邻两标记之间的距离范围是0~14.9 cM。NBS标记分布在8个连锁群中, 这些标记大部分聚在一起。本研究为今后的基因定位及重要农艺性状的分析提供框架图。此外, 研究NBS profiling 方法在花椰菜中的稳定性和有效性以及NBS-LRR类RGA在花椰菜基因组中的分布和特点。     

A chromosome region-specific mapping strategy reveals gene-rich telomeric ends in wheat

A strategy is described for rapid chromosome region-specific mapping in hexaploid wheat (Triticum aestivum L. em. Thell., 2n=6x=42, AABBDD). The method involves allocation of markers to specific chromosome regions by deletion mapping and ordering of probes by high resolution genetic mapping in Triticum tauschii, the D-genome progenitor species. The strategy is demonstrated using 26 chromosome deletion lines for wheat homoeologous group-6. Twenty-five DNA probes from the T. tauschii genetic linkage map and six wheat homoeologous group-6 specific probes were mapped on the deletion lines. Twenty-four of the 25 probes from 6D of T. tauschii also mapped on wheat homoeologous group-6 chromosomes, and their linear order in wheat is the same as in T. tauschii. A consensus physical map of wheat group-6 was constructed because the linear order and the relative position of the probe loci was the same among the three group-6 chromosomes. Comparison of the consensus physical map with the genetic map demonstrated that most of the recombination occurs in the distal ends of the wheat chromosomes. Most of the loci mapped in the distal regions of the chromosomes. The probes were mostly either PstI genomic clones or cDNA clones indicating that the undermethylated single-copy sequences are concentrated in the distal ends of the wheat chromosomes. Fifteen loci are uniformly distributed in the distal 11% of the group-6 chromosomes. Physically, the region spans only 0.58 m, which in wheat translates to about 40 Mb of DNA. The average distance between the markers is, therefore, less than 2.7 Mb and is in the range of PFGE (pulsed-field gel electrophoresis) resolution. Any gene present in the region can be genetically ordered with respect to the markers since the average recombination frequency in the region is very high (>90 cM genetic distance).     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

A microsatellite marker based linkage map of tobacco

We report the first linkage map of tobacco (Nicotiana tabacum L.) generated through microsatellite markers. The microsatellite markers were predominantly derived from genomic sequences of the Tobacco Genome Initiative (TGI) through bioinformatics screening for microsatellite motives. A total of 684 primer pairs were screened for functionality in a panel of 16 tobacco lines. Of those, 637 primer pairs were functional. Potential parents for mapping populations were evaluated for their polymorphism level through genetic similarity analysis. The similarity analysis revealed that the known groups of tobacco varieties (Burley, Flue-cured, Oriental and Dark) form distinct clusters. A mapping population, based on a cross between varieties Hicks Broad Leaf and Red Russian, and consisting of 186 F2 individuals, was selected for mapping. A total of 282 functional microsatellite markers were polymorphic in this population and 293 loci could be mapped together with the morphological trait flower color. Twenty-four tentative linkage groups spanning 1,920 cM could be identified. This map will provide the basis for the genetic mapping of traits in tobacco and for further analyses of the tobacco genome. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.