Inheritance of seed color and molecular markers linked to the seed color gene in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Seed color inheritance in Brassica juncea was studied in F₁, F₂ and BC₁ populations. Seed color was found under the control of the maternal genotype, and the brown-seeded trait was dominant over the yellow-seeded trait. Segregation analysis revealed that one pair of major genes controlled the seed coat color. To develop markers linked to the seed color gene, AFLP (amplified fragments length polymorphism) combined with BSA (bulk segregant analysis) technology was used to screen the parents and bulks selected randomly from an F₂ population (Wuqi yellow mustard × Wugong mustard) consisting of 346 individuals. From a survey of 512 AFLP primer combinations, 15 AFLP markers located on either side of the gene were identified, and the average distance between markers was 2.59 cM. P11MG15 was a cosegregated marker, and the closest markers (P03MC08, P16MC02 and P11MG01) were at a distance of 0.3, 0.3 and 0.7 cM from the target gene, respectively. In order to utilize the markers for breeding of yellow-seeded varieties, four AFLP markers, P11MG01, P15MG15, P09MC12 and P16MC02 were successfully converted into SCAR (sequence characterized amplified region) markers. The seed color trait controlled by the single gene together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded varieties. The markers found in the present study could accelerate the step of map-based cloning of the target gene.     

Hybridisation and introgression between Brassica napus and B. rapa in the Netherlands

We used flow cytometry, chromosome counting and AFLP markers to investigate gene flow from the crop plant oilseed rape, Brassica napus (AACC) to wild B. rapa (AA) in the Netherlands. From 89 B. napus source populations investigated, all near cropping fields or at transhipment sites, only 19 contained a B. rapa population within a 2.5‐km radius. During our survey we found only three populations with F1 hybrids (AAC), as recognized by their nine extra chromosomes and by flow cytometry. These hybrids were all collected in mixed populations where the two species grew in close proximity. Populations with F1 hybrids were not close to crops, but instead were located on road verges with highly disturbed soils, in which both species were probably recruited from the soil seed bank. Many plants in the F2, BC1 or higher backcrosses are expected to carry one to eight C chromosomes. However, these plants were not observed among the hybrids. We further investigated introgression with molecular markers (AFLP) and compared sympatric B. rapa populations (near populations of B. napus) with control populations of B. rapa (no B. napus within at least 7 km). We found no difference between sympatric and control populations in the number of C markers in B. rapa, nor did we find that these sympatric populations closely resembled B. napus. Our data show that hybrids occur but also suggest no recent introgression of alleles from the crop plant B. napus into wild B. rapa in the Dutch populations studied.     

Genetic and physical fine mapping of a multilocular gene Bjln1 in Brassica juncea to a 208-kb region

Most of the germplasm resources in Brassica juncea produce silique with only two locules, whereas a few varieties can produce silique with three or four locules. The increase in locule number in B. juncea has been shown to cause an increase in the number of seeds per silique, resulting in an increase in the yield per plant. Thus, the development of high-locule-number varieties may be an effective way of improving the yield of B. juncea. Duoshi, a B. juncea landrace originating from the Qinghai–Tibetan plateau, produces silique with 3–4 locules. Genetic analysis has shown that the high-locule-number trait in Duoshi is determined by two recessive genes, tentatively designated as Bjln1 and Bjln2. For fine mapping of the Bjln1 gene, a BC3 population was developed from the cross between Duoshi (multilocular parent) and Xinjie (bilocular parent). Using a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis, only two AFLP markers linked to Bjln1 were identified. Preliminary linkage analysis showed that the two AFLP markers were located on the same side of Bjln1. Blast analysis revealed that the sequences of the two AFLP markers had homologues on Scaffold000019 at the bottom of B. rapa A7. Using the results of linkage analysis and BlastN searches, simple sequence repeat (SSR) markers were subsequently developed based on the sequence information from B. rapa A7. Seven SSR markers were eventually identified, of which ln 8 was co-segregated with Bjln1. ln 7 and ln 9, the closest flanking markers, were mapped at 2.0 and 0.4 cM distant from the Bjln1 gene, respectively. The SSR markers were cloned, sequenced and mapped on A7 of B. rapa (corresponding to J7 in the A genome of B. juncea). The two closest flanking markers, ln 7 and ln 9, were mapped within a 208-kb genomic region on B. rapa A7, in which the Bjln1 gene might be included. The present study may facilitate cloning of the Bjln1 gene as well as the selection process for developing multilocular varieties in B. juncea by marker-assisted selection and genetic engineering.     

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B. nigra mitochondrial main circle genome with 232,407 bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B. rapa and B. oleracea had a better syntenic relationship than B. nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B. napus, in which napus‐type B. napus was derived from B. oleracea, while polima‐type B. napus was inherited from B. rapa. In addition, the mitochondrial genome of napus‐type B. napus was closer to botrytis‐type than capitata‐type B. oleracea. The sub‐stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.     

Genome-Wide Analysis of Seed Acid Detergent Lignin (ADL) and Hull Content in Rapeseed (Brassica napus L.)

A stable yellow-seeded variety is the breeding goal for obtaining the ideal rapeseed (Brassica napus L.) plant, and the amount of acid detergent lignin (ADL) in the seeds and the hull content (HC) are often used as yellow-seeded rapeseed screening indices. In this study, a genome-wide association analysis of 520 accessions was performed using the Q + K model with a total of 31,839 single-nucleotide polymorphism (SNP) sites. As a result, three significant associations on the B. napus chromosomes A05, A09, and C05 were detected for seed ADL content. The peak SNPs were within 9.27, 14.22, and 20.86 kb of the key genes BnaA.PAL4, BnaA.CAD2/BnaA.CAD3, and BnaC.CCR1, respectively. Further analyses were performed on the major locus of A05, which was also detected in the seed HC examination. A comparison of our genome-wide association study (GWAS) results and previous linkage mappings revealed a common chromosomal region on A09, which indicates that GWAS can be used as a powerful complementary strategy for dissecting complex traits in B. napus. Genomic selection (GS) utilizing the significant SNP markers based on the GWAS results exhibited increased predictive ability, indicating that the predictive ability of a given model can be substantially improved by using GWAS and GS.     

Fine mapping of the recessive genic male sterility gene (<Emphasis Type="Italic">Bnms3</Emphasis>) in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The Brassica napus oilseed rape line, 7-7365AB, is a recessive epistatic genic male sterile (RGMS) two-type line system. The sterility is controlled by two pairs of recessive duplicate genes (Bnms3 and Bnms4) and one pair of recessive epistatic inhibitor gene (Bnrf). Homozygosity at the Bnrf locus (Bnrfrf) inhibits the expression of the two recessive male sterility genes in homozygous Bnms3ms3ms4ms4 plants and produces a male fertile phenotype. This line has a good potential for heterosis utilization but it is difficult to breed heterotic hybrids without molecular markers. To develop markers linked to the BnMs3 gene, amplified fragment length polymorphism (AFLP) technology was applied to screen the bulks of sterile and fertile individuals selected randomly from a population of near-isogenic lines (NIL) consisting of 2,000 plants. From a survey of 1,024 primer combinations, we identified 17 AFLP markers linked to the BnMs3 gene. By integrating the previous markers linked to the BnMs3 gene into the genetic map of the NIL population, two markers, EA01MC12 and EA09P06, were located on either side of the BnMs3 gene at a distance of 0.1 and 0.3 cM, respectively. In order to use the markers for male sterile line breeding, five AFLP markers, P05MG05, P03MG04, P11MG02, P05MC11₂₅₀, and EA09P06, were successfully converted into sequence characterized amplified region (SCAR) markers. Two of these, P06MG04 and sR12384, were subsequently mapped on to linkage group N19 using two doubled-haploid mapping populations available at our laboratory derived from the crosses Tapidor × Ningyou7 and Quantum × No2127-17. The markers found in the present study should improve our knowledge of recessive genic male sterility (RGMS), and accelerate the development of male sterile line breeding and map-based cloning.     

Origins of the amphiploid species <Emphasis Type="Italic">Brassica napus</Emphasis>L. investigated by chloroplast and nuclear molecular markers

Background  

The amphiploid species Brassica napus (oilseed rape, Canola) is a globally important oil crop yielding food, biofuels and industrial compounds such as lubricants and surfactants. Identification of the likely ancestors of each of the two genomes (designated A and C) found in B. napus would facilitate incorporation of novel alleles from the wider Brassica genepool in oilseed rape crop genetic improvement programmes. Knowledge of the closest extant relatives of the genotypes involved in the initial formation of B. napus would also allow further investigation of the genetic factors required for the formation of a stable amphiploid and permit the more efficient creation of fully fertile re-synthesised B. napus. We have used a combination of chloroplast and nuclear genetic markers to investigate the closest extant relatives of the original maternal progenitors of B. napus. This was based on a comprehensive sampling of the relevant genepools, including 83 accessions of A genome B. rapa L. (both wild and cultivated types), 94 accessions of B. napus and 181 accessions of C genome wild and cultivated B. oleracea L. and related species.     

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T₁ single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC₁F₁, BC₂F₁) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC₂F₂ Bulk) were generated by crossing BC₂F₁ individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC₂F₂ Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F₁ hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC₂F₂ Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC₂F₂ Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F₁, BC₁F₁ and BC₂F₁). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.Communicated by J. Dvorak     

RFLP and AFLP Analysis of Inter- and Intraspecific Variation of Brassica rapa and B. napus Shows that B. rapa Is an Important Genetic Resource for B. napus Improvement

Fingerprinting of 29 accessions of oilseed rape, including seven accessions of Brassica napus, and 22 accessions of B. rapa (B. campestris) from Europe, North America, and China was analyzed using RFLP and AFLP markers. In total, 1 477 polymorphic RFLP bands and 183 polymorphic AFLP bands from 166 enzyme-probe combinations and two pairs of AFLP primers, respectively, were scored for the 29 accessions. On average, RFLP analysis showed that the Arabidopsis EST probe detected more polymorphic bands in Brassica than the random genomic probe performed. More polymorphic RFLP markers were detected with the digestion of EcoR I or BamH I than HindIII. According to the number of bands amplified from each accession, the copy numbers of each gene in the genomes of B. rapa and B. napus were estimated. The average copy numbers in B. rapa of China, B. rapa of Europe, and B. napus, were 3.2, 3.1, and 2.9, respectively. Genetic distance based on the AFLP data was well correlated with that based on the RFLP data (r = 0.72, P<0.001), but 0.39 smaller on average. Genetic diversity analysis showed that Chinese B. rapa was more polymorphic than Chinese B. napus and European materials. Some European B. napus accessions were clustered into European B. rapa, which were distinctly different from Chinese B. napus. The larger variations of Chinese accessions of B. rapa suggest that they are valuable in oilseed rape breeding. Novel strategies to use intersubgenomic heterosis between genome of B. rapa (A^rA^r) and genome of B. napus (AⁿAⁿCⁿCⁿ) were elucidated.     

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 × Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 × Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10–70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

QTL Mapping of Seed Coat Color for Yellow Seeded Brassica napus

The development of yellow-seeded varieties of Brassica napus for improving the oilseed quality characteristics of lower fiber content and higher protein and oil content has been a major focus of breeding researches worldwide in recent years. With the black-seeded ‘Youyan 2’ as male and the yellow-seeded GH₀₆ as female parents respectively, F₂ population of 132 individuals were obtained. A linkage map was constructed with 164 markers including 125 AFLP, 37 SSR, 1 RAPD and 1 SCAR markers distributed over 19 linkage groups covering approximately 2 549.8 cM with an average spacing of 15.55 cM. Two loci located on the 5th and 19th group were detected for the trait of seed coat color based on the linkage group using multiple interval mapping method and explained 46% and 30.9% of the phenotypic variation, respectively.     

Fine mapping of a dominant thermo-sensitive genic male sterility gene (BntsMs) in rapeseed (Brassica napus) with AFLP- and Brassica rapa-derived PCR markers

Key message

A new thermo-sensitive dominant genic male sterility (TSDGMS) line of Brassica napus was found and mapped in this paper. Our result will greatly accelerate the map-based cloning of the BntsMs gene.

Abstract

TE5A is a thermo-sensitive dominant genic male sterility line originating from spontaneous mutation of the inbred line TE5 in Brassica napus and provides a promising system for the development of hybrid cultivars. Genetic analysis has revealed that the BntsMs mutant is controlled by a single, dominant gene. Here, we describe the fine mapping of BntsMs using amplified fragment length polymorphism (AFLP) and intron polymorphism (IP) methodologies. We screened 1,024 primer combinations and then identified five AFLP markers linked to the BntsMs gene, two of which were successfully converted into sequence-characterised amplified region (SCAR) markers. The linkage of the markers was identified by analysing a large BC₂ population of 700 recessive-fertility individuals. Two SCAR markers were found in the flanking region of the BntsMs gene at distance of 3.5 and 4.8 cm. Based on sequence information from the previously screened AFLP markers and on genome organisation comparisons of the A genome of Brassica rapa and Arabidopsis, seven IP markers linked to the BntsMs gene were developed. By analysing the 700 recessive-fertility individuals, two IP markers, IP004 and IP470, were localised to the flanking region of the BntsMs gene at a distance of 0.3 and 0.2 cm, respectively. A comparison of the B. rapa and Arabidopsis genomes revealed 27 genes of B. rapa in the flanking region of these two IP markers. It is likely that the molecular markers developed from these investigations will greatly accelerate the positional cloning of the BntsMs gene.     

Maternal Inheritance of Chloroplasts between <Emphasis Type="Italic">Brassica rapa</Emphasis> and F<Subscript>1</Subscript>-hybrids Demonstrated by cpDNA Markers Specific to Oilseed Rape and <Emphasis Type="Italic">B. rapa</Emphasis>

Controlled reciprocal crosses between B. rapa and F₁-hybrids (B. napus (♀) × B. rapa), giving 20 pair-crossings, were made to reveal possible irregularities in chloroplast inheritance during production of BC₁s. Despite the close relationship of chloroplasts in B. rapa and B. napus, development of PCR-based molecular markers specific to B. rapa chloroplasts and B. napus chloroplasts was successful. Offspring from each cross were investigated and among these, we found no irregular chloroplast inheritance, since their plastid genotypes in all cases were identical to that of their mother. With a certainty of 95% our data indicate that the probability that chloroplasts are being inherited paternally is less than 0.015. In oilseed rape, pollen-mediated transgene-dispersal poses a well-known risk. Our results support development of transplastomic oilseed rape as an approach to reduce transgene dispersal.     

The reference genetic linkage map for the multinational Brassica rapa genome sequencing project

We describe the construction of a reference genetic linkage map for the Brassica A genome, which will form the backbone for anchoring sequence contigs for the Multinational Brassica rapa Genome Sequencing Project. Seventy-eight doubled haploid lines derived from anther culture of the F₁ of a cross between two diverse Chinese cabbage (B. rapa ssp. pekinensis) inbred lines, ‘Chiifu-401-42’ (C) and ‘Kenshin-402-43’ (K) were used to construct the map. The map comprises a total of 556 markers, including 278 AFLP, 235 SSR, 25 RAPD and 18 ESTP, STS and CAPS markers. Ten linkage groups were identified and designated as R1–R10 through alignment and orientation using SSR markers in common with existing B. napus reference linkage maps. The total length of the linkage map was 1,182 cM with an average interval of 2.83 cM between adjacent loci. The length of linkage groups ranged from 81 to 161 cM for R04 and R06, respectively. The use of 235 SSR markers allowed us to align the A-genome chromosomes of B. napus with those of B. rapa ssp. pekinensis. The development of this map is vital to the integration of genome sequence and genetic information and will enable the international research community to share resources and data for the improvement of B. rapa and other cultivated Brassica species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.