CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.)

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line ’Vedrantais’ were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible (’Vedrantais’) lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F₂ individuals from crosses of ’Vedrantais’ x PI 161375 and ’Ananas Yokneam’×MR-1 respectively, and 17 families from a backcross BC₁S₁ population derived from the breeding line ’MD8654’ as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F₂ individuals and BC₁S₁ families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived. Received: 28 July 1998 / Accepted: 7 December 1998     

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei. Received: 16 April 1999 / Accepted: 13 August 1999     

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.     

A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers

The silkworm, Bombyx mori is a beneficial insect of great economic importance in China for its silk production. In this study, we obtained 11 cleaved amplified polymorphic sequence (CAPS) markers and one PCR polymorphism marker from the genes of the silkworm, B. mori. A backcross progeny analysis showed that all these molecular markers were segregated in a Mendelian fashion and that polymorphisms were co-dominant. These markers were used to investigate the genetic diversity among 29 strains of B. mori from China, Japan and Europe. Cluster analysis, based on the genetic similarities calculated from CAPS data, grouped these strains roughly according to their geographical origin. One group contained silkworm strains from Europe and some of the Japanese strains were interspersed into the Chinese groups, whereas other Japanese strains clustered together.     

Multiplex-endonuclease genotyping approach (mega): a tool for the fine-scale detection of unlinked polymorphic DNA markers

Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most detailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of polymorphisms at one or two restriction sites. To combine increased discriminatory power with the stringency of polymerase chain reaction amplification, it would be beneficial to access additional independent restriction sites per analysis, and to amplify subsets of DNA restriction fragments with only one pair of oligonucleotide primers. We have now developed a unique approach that permits the simultaneous use of four or more endonucleases in combination with one pair of adapters/primers, and applied it to genotype 21 trypanosome populations to subspecific level. The approach takes advantage of the fact that some endonucleases create cohesive ends that are compatible with the overhang sites created by other endonucleases. We demonstrate the greater resolution of identifiable polymorphic fragments over the conventional ligation-mediated restriction analysis method, and discuss the value of the approach as a tool for fine genetic mapping of Trypanosoma brucei. Finally, we propose use of the method for fine characterisation and for identifying co-dominant genetic markers in a variety of other taxa. Edited by: W. HennigAn erratum to this article can be found at     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

AFLP and CAPS linkage maps of Cryptomeria japonica

We have used two DNA marker systems, AFLP and CAPS, in a two-way pseudo-testcross strategy applied to an F₁ population to construct genetic linkage maps of two local sugi cultivars. The AFLP markers detected about eight polymorphisms per parent per primer combination. Using 38 primer combinations, 612 AFLPs were detected in ’Haara 4’ and ’Kumotooshi’, of which 305 segregated in a 1:1 ratio (P>0.05). A total of 91 markers (83 AFLP and 8 CAPS) in ’Haara 4’ and 132 (123 AFLP and 9 CAPS) in ’Kumotooshi’ were distributed among 19 and 23 linkage groups, respectively, each of which included 2–17 markers. Maps of ’Haara 4’ and ’Kumotooshi’ spanned 1266.1 cM and 1992.3 cM, and covered approximately 50% and 80% of the sugi genome, respectively. Sequences derived from cDNA, which were previously used to construct a sugi linkage map, were also placed on our linkage maps as CAPS markers. Where a ’two-way pseudo-testcross’ is used, more than half of the sugi CAPS developed can be used to construct linkage maps for each parental family. The saturation of mapped markers, and the integration of several linkage maps derived from different mapping populations, is anticipated in the near future. Received: 15 August 1999 / Accepted: 27 August 1999     

A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)

A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

The construction of a genetic linkage map of red raspberry (Rubus idaeus subsp. idaeus) based on AFLPs, genomic-SSR and EST-SSR markers

Breeding in raspberry is time-consuming due to the highly heterozygous nature of this perennial fruit crop, coupled with relatively long periods of juvenility. The speed and precision of raspberry breeding can be improved by genetic linkage maps, thus facilitating the development of diagnostic markers for polygenic traits and the identification of genes controlling complex phenotypes. A genetic linkage map (789 cM) of the red raspberry Rubus idaeus has been constructed from a cross between two phenotypically different cultivars; the recent European cultivar Glen Moy and the older North American cultivar Latham. SSR markers were developed from both genomic and cDNA libraries from Glen Moy. These SSRs, together with AFLP markers, were utilised to create a linkage map. In order to test the utility of the genetic linkage map for QTL analysis, morphological data based on easily scoreable phenotypic traits were collected. The segregation of cane spininess, and the root sucker traits of density and spread from the mother plant, was quantified in two different environments. These traits were analysed for significant linkages to mapped markers using MapQTL and were found to be located on linkage group 2 for spines and group 8 for density and diameter. The availability of co-dominant markers allowed heterozygosities to be calculated for both cultivars.     

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.     

Testing Mendelian inheritance from field-collected parasites: Revealing duplicated loci enables correct inference of reproductive mode and mating system

Cryptic aspects of parasite population biology, e.g., mating systems, are increasingly being inferred from polymorphic and co-dominant genetic markers such as microsatellite loci. Underlying the use of such co-dominant markers is the assumption of Mendelian inheritance. The failure to meet this assumption can lead to artifactual statistics and erroneous population inferences. Here, we illustrate the importance of testing the Mendelian segregation and assortment of genetic markers and demonstrate how field-collected samples can be utilised for this purpose. To examine the reproductive mode and mating system of hermaphroditic parasites, we developed microsatellites for the cestode, Oochoristica javaensis. Among loci, we found a bimodal distribution of F_IS (a fixation index that quantifies the deviation from Hardy–Weinberg equilibrium within subpopulations) values where loci were either highly negative (close to −1) or highly positive (∼0.8). By conducting tests of Mendelian segregation from natural crosses, we determined that loci with negative F_IS values were in fact duplicated loci that were amplified by a single primer pair. Genetic crosses also provided linkage data and indicated that the duplicated loci most likely arose via tandem duplications rather than whole genome/chromosome duplications. By correcting for the duplicated loci, we were able to correctly infer that O. javaensis has sexual reproduction, but the mating system is highly inbred. To assist others in testing Mendelian segregation and independent assortment from natural samples, we discuss the benefits and limitations, and provide guidelines for particular parasite systems amenable to the methods employed here.     

Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones

 We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine (Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use. By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics. Received: 10 February 1998 / Accepted: 25 February 1998     

Construction of a genetic linkage map using simple sequence repeat markers from expressed sequence tags for cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta) is an economically important crop that is grown in tropical and sub-tropical regions. Use of molecular technology for genetic improvement of cassava has been limited by the lack of a large set of DNA markers and a genetic map. Therefore, the aims here were to develop additional simple sequence repeat (SSR) markers from the public expressed sequence tags (ESTs), and to construct a genetic linkage map. In this study, we designed 425 EST-SSR markers from sequences obtained from the cassava EST database in GenBank, and integrated them with 667 SSR markers from a microsatellite-enriched genomic sequence received from the International Center for Tropical Agriculture (CIAT). Of these, 107 EST-SSR and 500 genomic SSR primer pairs showed polymorphic patterns when screened in two cassava varieties, Hauy Bong 60 and Hanatee, which were used as female and male parental lines, respectively. Within the 107 and 500 primer pairs, 81 and 226 EST-SSR and SSR primer pairs were successfully genotyped with 100 samples of F₁ progeny, respectively. The results showed 20 linkage groups consisting of 211 markers—56 EST-SSR and 155 SSR markers—spanning 1,178 cM, with an average distance between markers of 5.6 cM and about 11 markers per linkage group. These novel EST-SSR markers provided genic PCR-based co-dominant markers that were useful, reliable and economical. The EST-SSRs were used together with SSR markers to construct the cassava genetic linkage map which will be useful for the identification of quantitative trait loci controlling the traits of interest in cassava breeding programs.     

Utility of RAPD markers in identifying genetic linkages to genes of economic interest in peach

The identification of molecular markers linked to economically important traits for use in crop improvement is very important in long-lived perennial species. Three-hundred-and-sixty RAPD primers were used with bulked segregant analysis to identify markers linked to loci of specific interest in peach [(Prunus persica) L. Batch] and peach x almond [(Prunus dulcis) Batch] crosses. The traits analyzed included flesh color, adhesion, and texture; pollen fertility; plant stature; and three isozyme loci. The Mendelian behavior of the RAPD loci was established, and RAPD markers were mapped relative to the loci controlling flesh color, adhesion, and texture, and the isozyme loci Mdh-1, 6Pgd-2 and Aat-1, as well as the existing RFLP genetic linkage map constructed previously using a peach x almond F₂ population. This technique has facilitated rapid identification of RAPD and RFLP markers that are linked to the traits under study. Loci controlling these traits mapped predominantly to linkage groups 2 and 3 of the peach genetic linkage map. Linkages to genes with both dominant and co-dominant alleles were identified, but linkages to dominant genes were more difficult to find. In several crosses, RAPD marker bands proved to be allelic. One co-dominant RAPD formed a heteroduplex band in heterozygous individuals and in mixtures of alternate homozygotes. The Mendelian behavior of the RAPD loci studied was established and the results suggest that RAPD markers will be useful for plant improvement in peach.     

Correlations between Genetic,Morphological, and Chemical Diversities in a Germplasm Collection of the Medicinal Plant Origanum vulgare L.

In total, 42 accessions of Origanum vulgare L., mostly originating from Europe, were evaluated, to detect molecular, quantitative morphological, and chemotype polymorphisms and to discover possible correlations between them. Twelve traits related to morphological characteristics were measured. The components in the essential oils were identified by GC/MS analysis, and the oil contents of 18 major compounds were determined. A total of 477 molecular polymorphisms including 214 AFLP (amplified fragment length polymorphism) and 263 SAMPL (selectively amplified microsatellite polymorphic loci) were used for genotyping. Euclidean distances of morphological and chemotypic data and genetic distances (1 – Dice's similarity) of molecular markers were compared by applying Mantel tests to ascertain the congruencies between them. A relatively high correlation between chemotypic patterns and genetic markers was identified, while a lower correlation was found between the morphological and genetic matrices. Pairwise analyses of correlation among all traits showed that the stem diameter was correlated to the essential‐oil yield and the carvacrol content. Cluster analysis, population inference, and principal component analysis revealed a broad genetic and chemical variation among the accessions. The knowledge of these diversities, found in this study, will allow a plant improvement of Origanum vulgare related to pharmaceutical and spice uses.     

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).     

Development of CAPS and dCAPS markers for <Emphasis Type="Italic">CYP82E4</Emphasis>, <Emphasis Type="Italic">CYP82E5v2</Emphasis> and <Emphasis Type="Italic">CYP82E10</Emphasis> gene mutants reducing nicotine to nornicotine conversion in tobacco

Nornicotine accumulation in tobacco is of concern because nornicotine is a precursor of N-nitrosonornicotine (NNN), a tobacco constituent recognized as a carcinogen by the health community. Nornicotine is derived from nicotine through a demethylation process catalyzed by nicotine demethylase enzymes. Three genes (CYP82E4, CYP82E5v2, and CYP82E10) have currently been identified that encode for these enzymes. Ethyl methane sulfonate has been used to introduce mutations into each of these genes to prevent production of functional gene products. These mutants represent a valuable tool for reducing nornicotine and NNN levels in cured tobacco leaves and their derived products. Methods are currently needed to rapidly and efficiently develop new cultivars possessing these mutant alleles. The objective of this study was to develop efficient, user-friendly DNA markers to identify these mutations based on single nucleotide polymorphisms (SNPs). Four dCAPS (derived cleaved amplified polymorphic sequence) markers were designed for a truncation mutation in CYP82E4, and a single marker was developed for a similar mutation in CYP82E5v2. Two CAPS (cleaved amplified polymorphic sequence) markers were designed for a missense mutation in CYP82E10. Because of the co-dominant nature of the CAPS and dCAPS markers, heterozygous and homozygous plants can be easily differentiated. Genotypes determined by the CAPS and dCAPS marker methods were validated by DNA sequencing and phenotypic analysis of plants carrying various mutant combinations. These markers can be used in marker-assisted selection programs to quickly introgress the desired mutations into commercial varieties in order to reduce nornicotine and NNN levels in tobacco leaves.