STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000     

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled-haploid progeny

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.     

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers

It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.     

Selection of stable Brassica napus-Brassica juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 2. A ‘to and fro’ strategy to localise and characterise interspecific introgressions on the B. napus genome

 A new strategy to localise and characterise interspecific introgressions in the genus Brassica is presented. It consists of the localisation of RAPD specific markers from the donor species (B. juncea) by RFLP on a genetic map of the recipient (B. napus) and on the observation of the disappearance of rapeseed markers in recombinant lines. With this method, we localised an interspecific introgression of B. juncea, which confers blackleg resistance at the cotyledon stage in B. napus, on the linkage group DY17 of the previously determined B. napus genetic map. The estimated size of the substituted B. napus fragment was 39 cM, and the resistance gene was introgressed into the rapeseed genome by homologous recombination. The significance of the different strategies used and the implication of these results in breeding programs are discussed. Received: 23 August 1997 / Accepted: 13 October 1997     

Molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus

We mapped the dwarf Bzh gene in B. napus with RAPD and RFLP markers. Research of the linked markers proceeded in two ways: a random approach through the construction of a detailed genetic map and targeting of the dwarf gene using both near-isogenic lines (NILs) and the bulked segregant analysis (BSA) method. The BSA approach was the most efficient in finding DNA markers linked to Bzh, whereas the efficiency of the NILs approach was limited by a too great similarity of the genetic background between the dwarf donor parent and the recurrent lines. Eight RAPD markers were identified as linked to Bzh, the closest being at 0.8±0.7 cM. The random genetic mapping approach added markers and extended the linkage group containing Bzh. This work represents the first step towards a better understanding of the dwarf mutation, the development of marker-assisted selection, and the cloning of the underlying gene responsible for dwarfing.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.)

The effectiveness of RFLP, DAMD-PCR, ISSR and RAPD markers in assessing polymorphism and relationships between 24 commercial lines of Phaseolus vulgaris L.was evaluated. We have used a Phaseolus-specific minisatellite sequence as a probe, which enabled 23 of the bean lines tested to be fingerprinted. Based on the sequence information obtained, primers corresponding to the bean-specific minisatellite core sequence were used in subsequent PCR amplifications. Our observations indicated that while the DAMD-PCR was sensitive in detecting genetic variation between bean species and between accessions of P. vulgaris, when used alone it may be limited in its ability to detect genetic variation among cultivated bean lines due to the low number of loci amplified. Only one out of the five ISSR primers tested was efficient in generating multiple band profiles, which was insufficient to distinguish all the different bean lines. Reproducible RAPD profiles were obtained, and these allowed us to differentiate all the genotypes tested with seven primers. We ultimately used only results from RFLP and RAPD markers to explore the genetic diversity among commercial bean lines. Both analyses led to the same clustering of the bean lines according to their geographical origins (United States or Europe). With respect to the European lines, the results obtained from RAPD data also enable the lines to be clustered according to their creators. Received: 15 January 2000 / Accepted: 21 March 2000     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.Abbreviations CrGC Crucifer Genetics Cooperative, University of Wisconsin-Madison - MES 1-morpholino-ethane sulfonate - MS-3,0 Murashige and Skoog medium containing 3% sucrose and no growth regulators - RAPD random amplified polymorphic DNA - RC rapid cycling - RFLP restriction fragment length polymorphism - std standard deviation - TE 10mM Tris, 1 mM EDTA, pH 8     

Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers

The three B genomes of Brassica contained in B. nigra, B. carinata and B. juncea were dissected by addition in B. napus. Using phenotypic, isozyme and molecular markers we characterized 8 alien B-genome chromosomes from B. nigra and B. carinata and 7 from B. juncea by constructing synteney groups. The alien chromosomes of the three different sources showed extensive intragenomic recombinations that were detected by the presence of the same loci in more than one synteny group but flanked by different markers. In addition, intergenomic recombinations were observed. These were evident in euploid AACC plants of the rapeseed phenotype derived from the addition lines carrying a few markers from the B genome due to translocations and recombinations between non-homoeologous chromosomes. The high plasticity of the Brassica genomes may have been an powerful factor in directing their evolution by hybridization and amphiploidy.     

Genetic relationships among melon breeding lines revealed by RAPD markers and agronomic traits

 RAPD markers and agronomic traits were used to determine the genetic relationships among 32 breeding lines of melon belonging to seven varietal types. Most of the breeding lines were Galia and Piel de Sapo genotypes, which are currently being used in breeding programmes to develop new hybrid combinations. A total of 115 polymorphic reliable bands from 43 primers and 24 agronomic traits were scored for genetic distance calculations and cluster analysis. A high concordance between RAPDs and agronomic traits was observed when genetic relationships among lines were assessed. In addition, RAPD data were highly correlated with the pedigree information already known for the lines and revealed the existence of two clusters for each varietal type that comprised the lines sharing similar agronomic features. These groupings were consistent with the development of breeding programmes trying to generate two separate sets of parental lines for hybrid production. Nevertheless, the performance of certain hybrids indicated that RAPDs were more suitable markers than agronomic traits in predicting genetic distance among the breeding lines analysed. The employment of RAPDs as molecular markers both in germplasm management and improvement, as well as in the selection of parental lines for the development of new hybrid combinations, is discussed. Received: 25 July 1997 / Accepted: 6 October 1997     

Development of a SCAR (sequence characterised amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L.

 A SCAR (sequence characterised amplified region) has been developed for optimal tagging of the dwarf Bzh gene in Brassica napus. A RAPD marker named OPMO7-730 was previously found closely linked to the dwarf locus at 0.8±0.7 cM. The DNA band corresponding to this marker was cloned and sequenced, and specific PCR primers were designed. The PCR test allowed us to amplify the locus corresponding to OPM07-730. With a restriction endonuclease digest and optimal electrophoresis conditions, three allelic forms of this marker have been recovered on the 40 B. napus accessions tested. The usefullness of this marker in breeding dwarf rapeseed lines is discussed. Received: 20 April 1998 / Accepted: 29 April 1998     

A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]

 A genetic linkage map of Brassica juncea was constructed based on restriction fragment length polymorphism (RFLP) detected by anonymous cDNA markers from B. napus, using a segregating F₁-derived doubled haploid (DH) progeny from a cross between a canola-quality mustard line (J90-4317) and a high-oil-content mustard line (J90-2733). The RFLP probes consisted of 229 cDNA probes from B. napus and a B. napus tandem repeat sequence, RDA2. The map consisted of 343 marker loci arranged in 18 major linkage groups plus five small segments with two to five marker loci, covering a total map distance of 2073 cM. Twenty-four percent of the markers were dominant in nature. Sixty-two percent of the marker loci were duplicated, and the majority were involved in inter-linkage group duplications, illustrating that complex duplications and subsequent rearrangements occurred after allopolyploidy. Deviation from the Mendelian segregation ratio for a DH population was observed for 27% of the markers. Two-thirds of these markers with a skewed segregation were clustered in 6 linkage groups and two unassigned segments. The overall average marker interval of the B. juncea map reported here was 6.6 cM, which would provide a marker density satisfactory for efficient use of the map in breeding applications, such as tagging of important agronomic traits and marker-assisted selection. Received: 14 May 1996 / Accepted: 11 October 1996     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

An integrated genetic linkage map of pepper (Capsicum spp.)

An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INTMAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers (common markers in at least two individual maps) were used for map integration. Most anchor markers (265) were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.These authors contributed equally to the work described in this paper(e-mail: