Mapping loci controlling vernalization requirement in Brassica rapa

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F₂ population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F₃ lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.     

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     

Genetics of Clubroot Resistance in <Emphasis Type="Italic">Brassica</Emphasis> Species

Clubroot disease, caused by the obligate plant pathogen Plasmodiophora brassicae Wor., is one of the most economically important diseases affecting Brassica crops in the world. The genetic basis of clubroot resistance (CR) has been well studied in three economically important Brassica species: B. rapa, B. oleracea, and B. napus. In B. rapa, mainly in Chinese cabbage, one minor and seven major CR genes introduced from European fodder turnips have been identified. Mapping of these CR genes localized Crr1 on R8, Crr2 on R1, CRc on R2, and Crr4 on R6 linkage groups of Chinese cabbage. Genes Crr3, CRa, CRb, and CRk mapped to R3, but at two separate loci, CRa and CRb are independent of Crr3 and CRk, which are closely linked. Further analysis suggested that Crr1, Crr2, and CRb have similar origins in the ancestral genome as in chromosome 4 of Arabidopsis thaliana. Genetic analysis of clubroot resistance genes in B. oleracea suggests that they are quantitative traits. Twenty-two quantitative trait loci (QTLs) were mapped in different linkage groups of B. oleracea. In B. napus, genetic analysis of clubroot resistance was found to be governed by one or two dominant genes, whereas resistance conferred by two recessive genes is reported. The quantitative analysis approach, however, proved that they are polygenic. In total, at least 16 QTLs have been detected on eight chromosomes of B. napus, N02, N03, N08, N09, N13, N15, N16, and N19. The chromosomal location of the other six QTLs is not clear. Cloning of any of these QTLs or resistance loci was not, however, possible until recently. Progress in genomics, particularly the techniques of comparative mapping and genome sequencing, supplements cloning and allows improved characterization of CR genes. Further development of DNA markers linked to CR genes will in turn hasten the breeding of clubroot-resistant Brassica cultivars.     

Comparative mapping of loci controlling winter survival and related traits in oilseed Brassica rapa and B. napus

Winter survival is an important characteristic of oilseedBrassica that is seeded in the fall in northern climates,and it may be affected by genetic variation for other cold-regulated traits,such as freezing tolerance and vernalization responsive flowering time. Weanalyzed immortalized populations of oilseed Brassica rapa(recombinant inbred lines) and B. napus (double haploidlines) derived from crosses of annual and biennial types in order to comparethe map positions and effects of quantitative trait loci controlling wintersurvival, nonacclimated and acclimated freezing tolerances, and flowering time.The B. napus population was evaluated in multiple winters,and six of the 16 total significant QTL for winter survival were detected inmore than one winter. Correspondence in the map positions of QTL controllingdifferent traits within species provided evidence that some alleles causinggreater acclimated freezing tolerance and later flowering time also contributedto increased winter survival. Correspondence in the map positions of QTLbetween species provided evidence for allelic variation at homologous loci inB. rapa and B. napus. The potentialrole of some candidate genes in regulating these traits is discussed.     

Mapping loci controlling flowering time in Brassica oleracea

The timing of the transition from vegetative to reproductive phase is a major determinant of the morphology and value of Brassica oleracea crops. Quantitative trait loci (QTLs) controlling flowering time in B. oleracea were mapped using restriction fragment length polymorphism (RFLP) loci and flowering data of F₃ families derived from a cabbage by broccoli cross. Plants were grown in the field, and a total of 15 surveys were made throughout the experiment at 5–15 day intervals, in which plants were inspected for the presence of flower buds or open flowers. The flowering traits used for data analysis were the proportion of annual plants (PF) within each F₃ family at the end of the experiment, and a flowering-time index (FT) that combined both qualitative (annual/biennial) and quantitative (days to flowering) information. Two QTLs on different linkage groups were found associated with both PF and FT and one additional QTL was found associated only with FT. When combined in a multi-locus model, all three QTLs explained 54.1% of the phenotypic variation in FT. Epistasis was found between two genomic regions associated with FT. Comparisons of map positions of QTLs in B. oleracea with those in B. napus and B. rapa provided no evidence for conservation of genomic regions associated with flowering time between these species.     

RFLP mapping of Brassica napus using doubled haploid lines

The combined use of doubled haploid lines and molecular markers can provide new genetic information for use in breeding programs. An F₁-derived doubled haploid (DH) population of Brassica napus obtained from a cross between an annual canola cultivar (Stellar) and a biennial rapeseed (Major) was used to construct a linkage map of 132 restriction fragment length polymorphism loci. The marker loci were arranged into 22 linkage groups and six pairs of linked loci covering 1016 cM. The DH map was compared to a partial map constructed with a common set of markers for an F₂ population derived from the same F₁ plant, and the overall maps were not significantly different. Comparisons of maps in Brassica species suggest that less recombination occurs in B. napus (n = 19) than expected from the combined map distances of the two hypothesized diploid progenitors, B. oleracea (n = 9) and B. rapa (n=10). A high percentage (32%) of segregating marker loci were duplicated in the DH map, and conserved linkage arrangements of some duplicated loci indicated possible intergenome homoeology in the amphidiploid or intragenome duplications from the diploid progenitors. Deviation from Mendelian segregation ratios (P < 0.05) was observed for 30% of the marker loci in the DH population and for 24% in the F₂ population. Deviation towards each parent occurred at equal frequencies in both populations and marker loci that showed deviation clustered in specific linkage groups. The DH lines and molecular marker map generated for this study can be used to map loci for agronomic traits segregating in this population. Present address Embrapa/Cenargen, C.P. 0.2372, CEP 70.770, Brasilia DF, Brazil     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Genetic analysis and mapping of genes controlling freezing tolerance in oilseedBrassica

Freezing tolerance is the ability of plants to survive subfreezing temperatures and is a major component of winter survival. In order to study the genetic regulation of freezing tolerance, an F2 population ofBrassica rapa and a doubled haploid population ofBrassica napus were assayedin vitro for relative freezing tolerance of acclimated and nonacclimated plants. Linkage maps developed previously were used to identify putative quantitative trait loci (QTL). Genomic regions with significant effects on freezing tolerance were not found for theB. napus population, but forB. rapa four regions were associated with acclimated freezing tolerance (FTA) and acclimation ability (FTB), and two unliked regions were associated with nonacclimated freezing tolerance (FTN). Acclimation ability was regulated by genes with very small additive effects and both positive and negative dominance effects. The allele from the winter parent at the FTN QTL had positive additive effects, but negative dominance effects. RFLP loci detected by a cold-induced and a stress-related cDNA fromArabidopsis thaliana mapped near two QTL for FTA/FTB. Further tests are needed to determine if alleles at these loci are responsible for the QTL effects we detected.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea

To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F₃ progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleracea CR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapa CR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.     

Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus

Oilseed rape (Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects. Received: 13 July 2001 / Accepted: 23 August 2001     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs mapping to nine different linkage groups of the B. rapa genome. Based on the RFLP data, phylogenetic trees were constructed using the PAUP microcomputer program. Within B. rapa, accessions of pak choi, narinosa, and Chinese cabbage from East Asia constituted a group distinct from turnip and wild European populations, consistent with the hypothesis that B. rapa had two centers of domestication. A wild B. rapa accession from India was positioned in the tree between European types and East Asian types, suggesting an evolutionary pathway from Europe to India, then to South China. Cultivated B. oleracea morphotypes showed monophyletic origin with wild B. oleracea or B. alboglabra as possible ancestors. Various kales constitute a highly diverse group, and represent the primitive morphotypes of cultivated B. oleracea from which cabbage, broccoli, cauliflower, etc. probably have evolved. Cauliflower was found to be closely related to broccoli, whereas cabbage was closely related to leafy kales. A great diversity existed among the 13 collections of nine-chromosome wild brassicas related to B. oleracea, representing various taxonomic states from subspecies to species. Results from these studies suggested that two basic evolutionary pathways exist for the diploid species examined. One pathway gave rise to B. fruticulosa, B. nigra, and Sinapis arvensis, with B. adpressa or a close relative as the initial ancestor. Another pathway gave rise to B. oleracea and B. rapa, with Diplotaxis erucoides or a close relative as the initial ancestor. Raphanus sativus and Eruca sativus represented intermediate types between the two lineages, and might have been derived from introgression or hybridization between species belonging to different lineages. Molecular evidence for an ascending order of chromosome numbers in the evolution of Brassica and allied genera was obtained on the basis of RFLP data and phylogenetic analysis.     

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.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary Restriction fragment length polymorphisms (RFLPs) of nuclear DNAs have been used to explore the origin and evolution of the six cultivated Brassica species. Extensive RFLP variation was found at the species, subspecies and variety levels. Based on RFLP data from Brassica and related genera, a detailed phylogenetic tree was generated using the PAUP microcomputer program, which permits a quantitative analysis of the interrelationships among Brassica species. The results suggested that 1) B. nigra originated from one evolutionary pathway with Sinapis arvensis or a close relative as the likely progenitor, whereas B. campestris and B. oleracea came from another pathway with a possible common ancestor in wild B. oleracea or a closely related nine chromosome species; 2) the amphidiploid species B. napus and B. juncea have evolved through different combinations of the diploid morphotypes and thus polyphyletic origins may be a common mechanism for the natural occurrence of amphidiploids in Brassica; 3) the cytoplasm has played an important role in the nuclear genome evolution of amphidiploid species when the parental diploid species contain highly differentiated cytoplasms. A scheme for the origins of diploid and amphidiploid species is depicted based on evidence gathered from nuclear RFLP analysis, cpDNA RFLP analysis, cytogenetic studies and classical taxonomy.     

Alignment of the conserved C genomes of Brassica oleracea and Brassica napus

A population of 169 microspore-derived doubled-haploid lines was produced from a highly polymorphic Brassica oleracea cross. A dense genetic linkage map of B. oleracea was then developed based on the segregation of 303 RFLP-defined loci. It is hoped that these lines will be used by other geneticists to facilitate the construction of a unified genetic map of B. oleracea. When the B. oleracea map was compared to one ofB. napus (Parkin et al. 1995), based on the same RFLP probes (Sharpe et al. 1995), good collinearity between the C-genome linkage groups of the two species was observed.     

RFLP mapping of loci controlling self-incompatibility in <Emphasis Type="Italic">Brassica campestris</Emphasis> and their comparative mapping with <Emphasis Type="Italic">B. napus</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

RFLP analysis of a cDNA probe SLG6, governing self incompatibility (SI) in Brassica oleracea, using a recombinant inbred population of Brassica campestris followed by genetic linkage analysis led to the detection of two marker loci, SLG6a and SLG6b controlling SI. SLG6a was mapped in linkage group (LG) 9 and was flanked by the RFLP markers ec4f10 (6.4 cM) and wg5b9 (4.2 cM). SLG6b positioned in LG 2 and was flanked by the RFLP markers wg2d11 (9.9 cM) and ec4e7 (26.9 cM). These results indicated the scope of marker-aided introgression of these genes into self-compatible genotypes for production of SI lines suitable for hybridization in B. campestris. Comparative mapping of LG 9 containing SLG6b with corresponding linkage groups of B. oleracea (BO 2) and B. napus (BN 16) led to the detection of small homologous regions with SLG6 locus linked with another RFLP locus. This evidenced for homology of the SLG genes across Brassica species and possibility of using any single cloned SLG gene for development of SI lines in any Brassica species.     

Development of public immortal mapping populations,molecular markers and linkage maps for rapid cycling <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

Publicly available genomic tools help researchers integrate information and make new discoveries. In this paper, we describe the development of immortal mapping populations of rapid cycling, self-compatible lines, molecular markers, and linkage maps for Brassica rapa and B. oleracea and make the data and germplasm available to the Brassica research community. The B. rapa population consists of 160 recombinant inbred (RI) lines derived from the cross of highly inbred lines of rapid cycling and yellow sarson B. rapa. The B. oleracea population consists of 155 double haploid (DH) lines derived from an F1 cross between two DH lines, rapid cycling and broccoli. A total of 120 RFLP probes, 146 SSR markers, and one phenotypic trait (flower color) were used to construct genetic linkage maps for both species. The B. rapa map consists of 224 molecular markers distributed along 10 linkage groups (A1–A10) with a total distance of 1125.3 cM and a marker density of 5.7 cM/marker. The B. oleracea genetic map consists of 279 molecular markers and one phenotypic marker distributed along nine linkage groups (C1–C9) with a total distance of 891.4 cM and a marker density of 3.2 cM/marker. A syntenic analysis with Arabidopsis thaliana identified collinear genomic blocks that are in agreement with previous studies, reinforcing the idea of conserved chromosomal regions across the Brassicaceae.     

Comparative and genetic studies of isozymes in resynthesized and cultivated Brassica napus L., B. campestris L. and B. alboglabra Bailey

Summary Enzyme electrophoresis was used to compare newly resynthesized Brassica napus with its actual parental diploid species, B. campestris and B. alboglabra. Comparisons were also made with cultivated B. napus. Of the eight enzyme systems assayed, four were monomorphic (hexokinase, malate dehydrogenase, mannose phosphate isomerase and peroxidase), whereas the remaining four were polymorphic (glucosephosphate isomerase, leucine aminopeptidase, phosphoglucomutase and shikimate dehydrogenase), when comparisons were made within or between species. The polymorphic enzyme patterns observed in the newly resynthesized B. napus disclosed that the homoeologous loci contributed by the parental species were expressed in the amphiploid. Analysis of the glucosephosphate isomerase enzyme in a breeding line (Sv 02372) of B. napus indicated that, in this case, the gene originating from B. campestris was switched off whereas that of B. oleracea was expressed. Duplicated enzyme loci were observed in B. campestris and B. alboglabra, thus providing additional evidence to support the hypothesis that these species are actually secondary polyploids derived from an unknown archetype of x=6.