The genetics of stamenoid petal production in oilseed rape (Brassica napus) and equivalent variation in Arabidopsis thaliana

 The agronomic potential of a Brassica napus variant with petalless flowers was compromised by an associated detrimental change in leaf morphology. Genetic analysis demonstrated the cosegregation of genes controlling both morphologies. Two STAP loci controlling the production of flowers with stamenoid petals were mapped to homoeologous locations in the genome of B. napus. The STAP loci were probably duplicate genes because they exhibited an epistatic interaction such that only plants homozygous for recessive stap alleles at both loci expressed the variant phenotype. The CURLY LEAF (CLF) gene of Arabidopsis thaliana pleiotropically influences both flower and leaf morphologies. The cloned CLF gene of Arabidopsis was homologous to a polymorphic B. napus locus coincident with one of the B. napus STAP loci. The possibility that CLF is a candidate gene for STAP suggests that the variant stap alleles of B. napus exert pleiotropic effects over both flower and leaf morphologies. Received: 26 August 1996 / Accepted: 20 September 1996     

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.     

Introgression of Brassica rapa subsp. sylvestris blackleg resistance into B. napus

Blackleg, caused by Leptosphaeria maculans, is one of the most economically important diseases of Brassica napus worldwide. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris (BRS) were previously identified. To transfer LepR1 and LepR2 from BRS into B. napus, interspecific hybridizations were made between the two species to form allotriploids. Analysis of microsatellite markers in two BC1 populations, WT3BC1 and WT4BC1, indicated that segregation fit a 1:1 ratio for BRS and non-BRS alleles on the A-genome linkage groups N2 and N10, the locations of LepR1 and LepR2, respectively. However, recombination frequencies in the allotriploid BC1 populations were at least twice those in the amphidiploid. The number of C-genome chromosomes in the BC1 plants was determined through marker analysis, which indicated averages of 5.9 and 5.0 per plant in the WT3BC1 and WT4BC1 populations, respectively. Two L. maculans isolates, WA51 and pl87-41, were used to differentiate plants carrying resistance genes LepR1 and LepR2. Surprisingly, only 4.0 and 16.6 % of the plants were resistant to isolates WA51 and pl87-41, respectively, in the WT3BC1 population, while 17.9 and 33.3 % of the plants were resistant to these isolates, respectively, in the WT4BC1 population. No association of resistance to isolate WA51 or pl87-41 with linkage group N2 or N10 was found. Based on cotyledon resistance and marker-assisted selection (MAS), BC1 plant WT4-4, which carried a resistance gene similar to LepR1, herein designated LepR1′, and BC2S1 plant WT3-21-25-9, which carried LepR2′, were identified. These plants were successively backcrossed with B. napus and MAS was employed in each generation to reduce non-resistance alleles associated with the BRS genome and to recover the full complement of C-genome chromosomes, resulting in highly blackleg-resistant B. napus lines.     

Genetic mapping of the <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> avirulence gene corresponding to the <Emphasis Type="Italic">LepR1</Emphasis> resistance gene of <Emphasis Type="Italic">Brassica napus</Emphasis>

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F₁ progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.     

Enhancing the carotenoid content of <Emphasis Type="Italic">Brassica napus</Emphasis> seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.     

Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris.

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus 'Surpass 400' are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. 'Surpass 400' also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of 'Surpass 400' was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on 'Surpass 400' was different from those observed on AD9 and MC1-8, indicating that 'Surpass 400' carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of 'Surpass 400' to most of the isolates tested were indistinguishable from those of AD49, which suggested 'Surpass 400' might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in 'Surpass 400'. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.     

Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape).

A genetic linkage map consisting of 399 RFLP-defined loci was generated from a cross between resynthesized Brassica napus (an interspecific B. rapa x B. oleracea hybrid) and "natural" oilseed rape. The majority of loci exhibited disomic inheritance of parental alleles demonstrating that B. rapa chromosomes were each pairing exclusively with recognisable A-genome homologues in B. napus and that B. oleracea chromosomes were pairing similarly with C-genome homologues. This behaviour identified the 10 A genome and 9 C genome linkage groups of B. napus and demonstrated that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of the amphidiploid species, B. napus. A range of unusual marker patterns, which could be explained by aneuploidy and nonreciprocal translocations, were observed in the mapping population. These chromosome abnormalities were probably caused by associations between homoeologous chromosomes at meiosis in the resynthesized parent and the F1 plant leading to nondisjunction and homoeologous recombination.     

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.     

用RFLP标记分析甘蓝型油菜的遗传多样性

以甘蓝型油菜的２８个基因组探针和两种限制性内切酶对包括４６个中国品种、９个欧洲品种在内的５９个甘蓝型油菜品种（系）的ＲＦＬＰ标记进行了分析。在放射自显影胶片上,共检测到４１０条具多态性的分子杂交带,表明甘蓝型油菜中存在着极为丰富的遗传变异。聚类分析结果表明,在相似性为４５％的水平上,可把中国甘蓝型油菜划分为６组：胜利油菜组、跃进油菜组、中油８２１组、远缘种质组、优质油菜组和变异不详组。欧洲冬油菜与以上６组存在着较显著的遗传距离。主成分分析的结果与上述分组较为一致。以上结果表明,对于扩大中国甘蓝型油菜的遗传基础,欧洲冬油菜无疑是一个重要的种质资源。另一方面,用典型的中国甘蓝型油菜与欧洲冬油菜配制的杂交种,较易产生强大的杂种优势。从对已进行了染色体定位的６１条放射自显影带的分析看,无论是上述分组内,还是分组间,ＲＦＬＰ的相对差异均主要表现在Ａ基因组中。讨论了致使Ａ基因组遗传变异较大的可能因素。     

Genetic analysis and genome mapping in Raphanus.

The first genetic map of the Raphanus genome was developed based on meiosis in a hybrid between Raphanus sativus (cultivated radish) and Raphanus raphanistrum (wild radish). This hybrid was used to produce a BC1 population of 54 individuals and an F2 population of 85 individuals. A total of 236 marker loci were assayed in these populations using a set of 144 informative Brassica RFLP probes previously used for genetic mapping in other crucifer species. The genetic maps derived from the BC1 and F2 populations were perfectly collinear and were integrated to produce a robust Raphanus map. Cytological observations demonstrated strict bivalent pairing in the R. sativus x R. raphanistrum hybrids. Productive pairing along the length of each chromosome was confirmed by the identification of nine extensive linkage groups and the lack of clustering of marker loci. Indeed, the distributions of both marker loci and crossovers was more random than those reported for other crop species. The genetic markers and the reference map of Raphanus will be of considerable value for future trait mapping and marker-assisted breeding in this crop, as well as in the intergenomic transfer of Raphanus genes into Brassica crops. The future benefits of comparative mapping with Arabidopsis and Brassica species are also discussed.