Linkage analysis of molecular markers and quantitative trait loci in populations of inbred backcross lines of Brassica napus L.

Backcross populations are often used to study quantitative trait loci (QTL) after they are initially discovered in balanced populations, such as F(2), BC(1), or recombinant inbreds. While the latter are more powerful for mapping marker loci, the former have the reduced background genetic variation necessary for more precise estimation of QTL effects. Many populations of inbred backcross lines (IBLs) have been developed in plant and animal systems to permit simultaneous study and dissection of quantitative genetic variation introgressed from one source to another. Such populations have a genetic structure that can be used for linkage estimation and discovery of QTL. In this study, four populations of IBLs of oilseed Brassica napus were developed and analyzed to map genomic regions from the donor parent (a winter-type cultivar) that affect agronomic traits in spring-type inbreds and hybrids. Restriction fragment length polymorphisms (RFLPs) identified among the IBLs were used to calculate two-point recombination fractions and LOD scores through grid searches. This information allowed the enrichment of a composite genetic map of B. napus with 72 new RFLP loci. The selfed and hybrid progenies of the IBLs were evaluated during two growing seasons for several agronomic traits. Both pedigree structure and map information were incorporated into the QTL analysis by using a regression approach. The number of QTL detected for each trait and the number of effective factors calculated by using biometrical methods were of similar magnitude. Populations of IBLs were shown to be valuable for both marker mapping and QTL analysis.

A bayesian approach to detect quantitative trait loci using Markov chain Monte Carlo.

Markov chain Monte Carlo (MCMC) techniques are applied to simultaneously identify multiple quantitative trait loci (QTL) and the magnitude of their effects. Using a Bayesian approach a multi-locus model is fit to quantitative trait and molecular marker data, instead of fitting one locus at a time. The phenotypic trait is modeled as a linear function of the additive and dominance effects of the unknown QTL genotypes. Inference summaries for the locations of the QTL and their effects are derived from the corresponding marginal posterior densities obtained by integrating the likelihood, rather than by optimizing the joint likelihood surface. This is done using MCMC by treating the unknown QTL, genotypes, and any missing marker genotypes, as augmented data and then by including these unknowns in the Markov chain cycle alone with the unknown parameters. Parameter estimates are obtained as means of the corresponding marginal posterior densities. High posterior density regions of the marginal densities are obtained as confidence regions. We examine flowering time data from double haploid progeny of Brassica napus to illustrate the proposed method.

Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana.

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans.

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.

Evaluation of Sclerotinia Stem Rot Resistance in Oilseed Brassica napus Using a Petiole Inoculation Technique Under Greenhouse Conditions.

A petiole inoculation technique was adapted for evaluating resistance of oilseed Brassica napus seedlings to Sclerotinia sclerotiorum. In the first of four experiments, four isolates of S. sclerotiorum were tested, two originating from soybean and two from B. napus. In all, 10 to 47 B. napus accessions were inoculated in the seedling stage and responses to isolates were evaluated using days to wilt (DW) and a lesion phenotype index (LP). There were no significant differences in virulence among the four isolates for DW and only slight differences for LP. However, significant differences (P < 0.0001) were observed among the B. napus accessions for DW and LP in this experiment and in subsequent experiments using one isolate. The responses of accessions were consistent among experiments and among evaluation criteria. Higher levels of resistance were found among winter-type than spring-type accessions, and among rapeseed-quality compared with canola-quality accessions. The most resistant accessions identified also were the most resistant when inoculated at the flowering stage. Terminal stems were inoculated immediately below the lowest flower and stem lesion length (SLL) was used to characterize the interaction phenotype of each accession. The petiole inoculation technique can be used successfully to differentiate oilseed B. napus germ plasm for response to S. sclerotiorum. This inoculation technique and the sources of resistance identified in this study may be used to determine inheritance resistance to S. sclerotiorum and for improving oilseed B. napus cultivars for resistance to this important pathogen.

Analyses of a multi-parent population derived from two diverse alfalfa germplasms: testcross evaluations and phenotype-DNA associations.

In a previous study, we showed that the genetic variation present in the Medicago sativa subsp. sativa Peruvian and M. sativa subsp. falcata WISFAL germplasms could be used to improve forage yields when favorable alleles were recombined and used in hybrid combination with cultivated alfalfa. In this paper, we present testcross forage yield and fall growth data for two seasons of a C0 population generated after intermating the Peruvian x WISFAL population for several generations. In addition, we conducted marker-trait association analysis as an attempt to identify Peruvian and WISFAL genomics regions affecting the targeted traits. Five and seven genomic regions were found significantly associated with forage yield and fall growth, respectively. In the case of fall growth, alleles from both accessions were positively associated with plant height. However, more alleles from WISFAL were positively associated with forage yield than from Peruvian. WISFAL is known for its winter hardiness and genomic regions with large effects on winter survival may have masked the effect of forage yield from Peruvian. The fact that most of the genomic regions discovered in this study have been previously associated with traits involved in winter hardiness validates our findings and suggests that associations between DNA fragments and agronomic traits can be detected without the necessity of developing bi-parental mapping populations.

Characterization of a dwarf gene in Brassica rapa, including the identification of a candidate gene.

Dwarf genes have been valuable for improving harvestable yield of several crop plants and may be useful in oilseed Brassica. We evaluated a dwarf gene, dwf2, from Brassica rapa in order to determine its phenotypic effects and genetic characteristics. The dwf2 mutant was insensitive to exogenous GA(3) for both plant height and flowering time, suggesting that it is not a mutation in the gibberellin biosynthesis pathway. The dwarf phenotype was controlled by a semidominant allele at a single locus. Near-isogenic lines that were homozygous or heterozygous for dwf2 had 47.4% or 30.0% reduction in plant height, respectively, compared to the tall wild-type line, and the reduction was due to reduced internode length and number of nodes. The dwf2 homozygous and heterozygous lines had the same or significantly higher numbers of primary branches than the wild-type line, but did not differ in flowering time. The DWF2 gene was mapped to the bottom of linkage group R6, in a region having homology to the top of Arabidopsis thaliana chromosome 2. The map position of DWF2 in comparison to markers in A. thaliana suggests it is a homolog of RGA ( repressor of ga1-3), which is a homolog of the wheat "Green Revolution" gene. This dwarf gene could be used to gain more insight on the gibberellin pathway and to reduce lodging problems in hybrid oilseed Brassica cultivars.

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus, B. oleracea, and Arabidopsis thaliana.

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms.

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs 'Triton' and 'Tower'. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.

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.