Marker-assisted selection of low erucic acid quantity in short duration Brassica rapa

Low erucic acid (LEA) rapeseed, which has accumulated mutant fatty acid elongase genes at the BnFAE1.1 and BnFAE1.2 loci of the A- and C-genome, respectively, is an important oilseed crop. Short growing turnip rape (B. rapa) is also important as a catch crop in the continuous cropping of rice in Asia but there is no LEA B. rapa cultivar for cultivation in South Asia. In order to develop LEA turnip rape cultivars, high erucic acid turnip rape cultivars were interspecifically crossed as recurrent parents to a canola quality rapeseed. In the meantime, we monitored incorporation of the mutant bnfae1.1 (e1) gene into A-genome of turnip rape, using a dCAPS primer pair, which can amplify PCR fragment only for the mutant e1 gene from A-genome. The early backcross progenies showed poor seed set, but which was improved in advanced progenies. Finally, homozygous e1e1 genotypes were established in the selfed progenies of BC₂–BC₃, and their LEA content was confirmed by gas-chromatography analysis. Our results and promising lines will contribute to LEA-trait selection in turnip rape and rapeseed breeding.     

Fatty acid composition of resynthesized Brassica napus and trigenomic Brassica void of genes for erucic acid in their A genomes

The fatty acid composition of seed oil of four interspecific hybrids, resulting from crosses between zero erucic acid Brassica rapa (AA), and high erucic acid Brassica alboglabra/Brassica oleracea (CC) and Brassica carinata (BBCC), void of erucic acid genes in their A‐genomes was examined. The erucic acid content in resynthesized Brassica napus (AACC) lines derived from these crosses was only about half that of the high erucic acid CC genome parents, indicating equal contributions of the two genomes to oil (fatty acid) synthesis and accumulation. The differences in C18 fatty acid synthesis between the parents were also evident in the resulting resynthesized B. napus plants. Hexaploid Brassica plants of the genomic constitution AABBCC, in which the AA genome was incapable of erucic acid synthesis, had lower erucic acid contents than the B. carinata (BBCC) parent. This is plausible considering the fact that the zero erucic acid AA genome contributes to oil synthesis in AABBCC plants, thus reducing erucic acid content.     

Molecular marker analysis of the linkage drag around the FAE1 loci of Brassica juncea during conventional backcross breeding

A post-transfer investigation was carried out to analyze the process of gene introgression in a conventional backcross-breeding program taking low erucic acid as target trait in Brassica juncea. FAE1 locus is involved in the elongation of oleic acid (C18:1) to erucic acid (C22:1). A high concentration of erucic acid in the seeds of Brassica species has been reported to be nutritionally undesirable. Molecular markers like AFLP markers, microsatellite markers, and gene-based SNP markers were used to determine the size of the donor parent chromosomal segments retained around the FAE1 genes in the individuals selected from different backcross generations. The genotype of the individuals was inferred from the genotype of the markers and the graphical genotypes were constructed using GGT software. Molecular marker analysis led to the identification of rare recombinants near the target locus with reduced size of introgressed segment from the donor parent. Based on the present study, we propose that marker-assisted backcross breeding in B. juncea could prove to be a promising tool for the transfer of many quality traits from unadapted East European germplasm to Indian cultivars.     

利用原位杂交及CAPS标记分析芸薹属A、B和C基因组间的关系

以来源于Brassica rapa基因组(AA)的重复序列(151 bp)为探针, 分别同二倍体白菜型油菜(AA, 2n=20)、甘蓝(CC, 2n=18)和异源四倍体芥菜型油菜(AABB, 2n=36)的中期染色体杂交, 白菜型油菜和甘蓝的所有染色体上都有杂交信号, 芥菜型油菜的染色体上显示出20个明显的信号, 其余染色体上信号很弱或无, 可以区分出A和B基因组。对来源于油菜3个基本种与3个复合种FAE1基因进行CAPS分析表明, 3个基本种表现出不同的酶切式样, 用Mbo I和Msp I酶切表现出多态性, 基因组A和C非常相似, 而基因组B与A、C关系较远, 同时3个复合种也并不是2个基本种的简单相加, 表明异源四倍体在长期进化过程中可能发生了重排和重组。     

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.     

Possibilities of direct introgression from Brassica napus to B. juncea and indirect introgression from B. napus to related Brassicaceae through B. juncea

The impact of genetically modified canola (Brassica napus) on biodiversity has been examined since its initial stage of commercialization. Various research groups have extensively investigated crossability and introgression among species of Brassicaceae. B. rapa and B. juncea are ranked first and second as the recipients of cross-pollination and introgression from B. napus, respectively. Crossability between B. napus and B. rapa has been examined, specifically in terms of introgression from B. napus to B. rapa, which is mainly considered a weed in America and European countries. On the other hand, knowledge on introgression from B. napus to B. juncea is insufficient, although B. juncea is recognized as the main Brassicaceae weed species in Asia. It is therefore essential to gather information regarding the direct introgression of B. napus into B. juncea and indirect introgression of B. napus into other species of Brassicaceae through B. juncea to evaluate the influence of genetically modified canola on biodiversity. We review information on crossability and introgression between B. juncea and other related Brassicaseae in this report.     

Erucic acid heredity in Brassica juncea - some additional information

Genetic studies were undertaken to reassess erucic acid heredity in Brassica juncea. Analysis of segregation in F₂ and BC₁ generations from two zero × high erucic acid crosses indicated that higher erucic acid in B. juncea was controlled by two dominant genes with additive effects, whereas segregation in a cross involving ‘CCWF 16′, a genotype having intermediate erucic acid (25.6%), and a zero erucic acid strain, indicated monogenic dominant control for intermediate erucic acid content. The B. juncea strain ‘CCWF 16’ was developed by hybridizing high‐erucic acid B. juncea cv.‘WF‐1’ with a ‘0’ erucic B. rapa cv.‘Candle’ followed by backcrossing with ‘WF‐1’ and half‐seed selection for low erucic acid in each backcross generation. This strategy resulted in substitution of the high erucic acid allele present in the A genome of B. juncea (AABB) by the zero erucic acid allele associated with ‘A’ genome of ‘Candle’. The intermediate erucic acid content in ‘CCWF 16’ was thus attributed to a gene present in the ‘BB’ genome. Experimental data clearly suggested that the gene (E₂) associated with the A genome had a greater contribution to the total erucic acid content in B. juncea than the gene (E₁) located on the B genome. This provided experimental evidence for a previous suggestion of unequal contributions of two dominant genes (E₁= 12%, E₂= 20%) to high erucic acid content in conventional digenomic Brassica species.     

芥菜型油菜FAE1基因序列特征及其与芥酸含量关系的初步分析

采用同源序列法对6个芥菜型油菜(Brassica juncea)品种(高芥酸、中芥酸、低芥酸)、2份白菜型油菜品种(高芥酸和低芥酸)和1份黑芥品种的FAE1基因进行克隆和测序表明,9个品种的FAE1基因编码区全长均为1522bp,不含内含子,均编码507个氨基酸残基。序列比较表明,芥菜型油菜中有两种FAE1基因序列(BjFAE1.1和BjFAE1.2),其亲缘种白菜型油菜和黑芥中各有一种FAE1基因序列(BrFAE1和BnFAE1),BjFAE1.1对应于白菜型油菜的BrFAE1,BjFAE1.2对应于黑芥的BnFAE1;BjFAE1.1和BjFAE1.2之间存在71bp处核苷酸变异和Hind III不同的酶切位点(第1415位和第1144位),蛋白质水平上存在15处氨基酸变异。比较不同芥酸含量品种的FAE1基因序列表明,BjFAE1.1基因存在2个SNP位点(第968位和第1265位),BjFAE1.2基因也有2个SNP位点(第49位和第237位),这4个SNP位点中有3个位点(第49位、第968位和第1265位)导致蛋白质水平上氨基酸的差异。其中BjFAE1.1基因第968位的碱基变化(C→T)引起的第323位氨基酸变化(Thr→Ile),能够解释芥菜型油菜和白菜型油菜高芥酸到低芥酸(中芥酸)的转变;第1265位的碱基变化(T→C)引起的第422位的氨基酸变化(Phe→Ser),能够部分解释芥菜型油菜的高芥酸到低芥酸(中芥酸)的转变,白菜型油菜的高芥酸和低芥酸品种在该位点的碱基没有变化。BjFAE1.2基因第49位的碱基变化(T→C)引起的第17位氨基酸的变化(Phe→Leu),可以解释芥菜型油菜的中芥酸变成高芥酸(低芥酸)。陕北黄芥低芥酸突变株1278-3的FAE1基因序列和国外低芥酸品种比较,只在第1265位出现变异。     

Assessing and broadening genetic diversity of a rapeseed germplasm collection

Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F₁ hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.     

Production of high yield short duration Brassica napus by interspecific hybridization between B. oleracea and B. rapa

Brassica napus is a leading oilseed crop throughout many parts of the world. It is well adapted to long day photoperiods, however, it does not adapt well to short day subtropical regions. Short duration B. napus plants were resynthesized through ovary culture from interspecific crosses in which B. rapa cultivars were reciprocally crossed with B. oleracea. From five different combinations, 17 hybrid plants were obtained in both directions. By self-pollinating the F₁ hybrids or introgressing them with cultivated B. napus, resynthesized (RS) F₃ and semi-resynthesized (SRS) F₂ generations were produced, respectively. In field trial in Bangladesh, the RS B. napus plants demonstrated variation in days to first flowering ranging from 29 to 73 days; some of which were similar to cultivated short duration B. napus, but not cultivated short duration B. rapa. The RS and SRS B. napus lines produced 2–4.6 and 1.6–3.7 times higher yields, respectively, as compared to cultivated short duration B. napus. Our developed RS lines may be useful for rapeseed breeding not only for subtropical regions, but also for areas such as Canada and Europe where spring rapeseed production can suffer from late spring frosts. Yield and earliness in RS lines are discussed.     

Long-term monitoring of feral genetically modified herbicide-tolerant Brassica napus populations around unloading Japanese ports

Genetically modified, herbicide-tolerant (GMHT) Brassica napus plants originating from seed spill have recently been found along roadsides leading from Japanese ports that unload oilseed rape. Such introductions have potential biodiversity effects (as defined by the Cartagena Protocol): these include replacement of native elements in the biota through competitive suppression or hybridization. We conducted surveys in the period 2006–2011 to assess such threats. We examined shifts in the population distribution and occurrence of GMHT plants in 1,029 volunteer introduced assemblages of B. napus, 1,169 of B. juncea, and 184 of B. rapa around 12 ports. GMHT B. napus was found around 10 of 12 ports, but its proportion in the populations varied greatly by year and location. Over the survey period, the distributions of a pure non-GMHT population around Tobata and a pure GMHT population around Hakata increased significantly. However, there was no common trend of population expansion or contraction around the 12 ports. Furthermore, we found no herbicide tolerant B. juncea and B. rapa plants derived from crosses with GMHT B. napus. Therefore, GMHT B. napus is not invading native vegetation surrounding its populations and not likely to cross with congeners in Japanese environment.     

Recent progress in drought and salt tolerance studies in Brassica crops

Water deficit imposed by either drought or salinity brings about severe growth retardation and yield loss of crops. Since Brassica crops are important contributors to total oilseed production, it is urgently needed to develop tolerant cultivars to ensure yields under such adverse conditions. There are various physiochemical mechanisms for dealing with drought and salinity in plants at different developmental stages. Accordingly, different indicators of tolerance to drought or salinity at the germination, seedling, flowering and mature stages have been developed and used for germplasm screening and selection in breeding practices. Classical genetic and modern genomic approaches coupled with precise phenotyping have boosted the unravelling of genes and metabolic pathways conferring drought or salt tolerance in crops. QTL mapping of drought and salt tolerance has provided several dozen target QTLs in Brassica and the closely related Arabidopsis. Many drought- or salt-tolerant genes have also been isolated, some of which have been confirmed to have great potential for genetic improvement of plant tolerance. It has been suggested that molecular breeding approaches, such as marker-assisted selection and gene transformation, that will enhance oil product security under a changing climate be integrated in the development of drought- and salt-tolerant Brassica crops.     

In silico-selection of Brassica rapa organelle genome-derived BACs using their end sequences and sequence level comparative analysis of the 124 kb mitochondrial genome sequences in the family Brassicaceae

We identified BAC clones which harbor DNAs derived from the B. rapa organelle genomes by in silico mapping of 80,292 B. rapa BAC end sequences on the Arabidopsis organelle genomes and subsequent insert size estimation and fingerprinting. A total of 1,048 putative chloroplast genome-derived BAC clones (2.6%) were identified. Fingerprinting and sequencing revealed that many of them represented the entire chloroplast genome (about 150 kb). Meanwhile, only 59 putative mitochondrial genome-derived BACs (0.15%) were identified and most of them showed rare agreement between the in silico map and fingerprinting. We sequenced BAC clone KBrB042G11 (42G11) and compared it to the mitochondrial genome of B. napus and A. thaliana which showed dynamic rearrangement events. The order of 33 orthologous genes was collinear between the 42G11 BAC and its counterpart in B. napus. Five distinctive rearrangements and two InDels were identified between these two closely related species and the rearrangements were related to the occurrence of small tandem repeat sequences. Sequences of the 33 orthologous genes in the homoeologous regions of B. napus and B. rapa were almost 100% identical. Gene orders showed no colinearity between Arabidopsis and Brassica even though 31 orthologous genes shared high sequence similarity with p-values over 1E-32. FISH analysis using the identified BAC revealed a large chloroplast genome insertion in the pericentromeric region of chromosome (chr.) 4 of B. rapa.     

Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

Development of synthetic Brassica napus lines for the analysis of “fixed heterosis” in allopolyploid plants

Summary Allopolyploids are widely spread in the plant kingdom. Their success might be explained by positive interactions between homoeologous genes on their different genomes, similar to the positive interactions between different alleles of one gene causing heterosis in heterozygous diploid genotypes. In allopolyploids, such interactions can also occur in homozygous genotypes, and may therefore be called “fixed heterosis”. As to our knowledge, no experimental data are available to support this hypothesis. We propose an experimental approach to quantify “fixed heterosis” in resynthesised Brassica napus and the detection of loci contributing to “fixed heterosis” via comparative QTL mapping in B. napus and its parental species B. rapa and B. oleracea. In order to develop a genetically balanced material, interspecific crosses between 21 Brassica rapa and 16 Brassica oleracea doubled haploid or inbred lines were performed. In total 3485 vital embryos have been obtained from 9514 pollinated buds. The success of interspecific hybridisation was highly depending on the maternal genotype (B. rapa) and ranged from 0 to 1.18 embryos per pollinated bud. For the genetic characterisation of the B. rapa and B. oleracea lines, a dendrogram was constructed based on 273 RAPD markers. Thus a well-characterised material is now available, which is suitable to analyse the effects of “fixed heterosis” and the interactions between homoeologous genes in allopolyploid species.     

Genetic diversity in advanced derivatives of Brassica interspecific hybrids

Genetic diversity among the 88 entries including eighty F₄ derivatives i.e., 20 each selected from Brassica crosses viz., B. juncea × B. napus, B. juncea × B. rapa var. toria, B. juncea ×B. rapa var. yellowsarson and B. tournefortii × B. juncea, and eight parent genotypes was assessed through multivariate analysis (D² statistic). Significant differences among the family groupsas well as within the family were recorded for all the 14 characters studied. The D² analysis revealed enormous diversity among the interspecific cross derivatives. The genetic distances calculated among different Brassica species revealed that B. tournefortii had maximumdiversity with B. juncea followed by B. napus, B.rapa var. toria and B. rapa var. yellow sarson.Amongst interspecific crosses, maximum diversity was noticed indescendants of cross B. tournefortii × B. juncea followed byB. juncea × B. napus, B. juncea × B.rapa var. toria and the least in the cross B. juncea ×B. rapa var. yellow sarson. These results indicated that the derivatives selected from cross of diverse parents revealed greater diversity. The clustering pattern showed that many derivatives of the cross fell into the same cluster but in many cases in spite of common ancestry many descendants of the cross spread over different clusters. The characters, namely, plant height, secondary branches per plant, days to flowering and1000-seed weight were contributed maximum towards genetic divergence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of bulked and redundant accessions of Brassica germplasm using assignment tests of microsatellite markers

This study was conducted to determine if Brassica germplasm bulks created and maintained by the USDA-ARS North Central Plant Introduction Station (NCRPIS) were made with genetically indistinguishable component accessions and to examine newly identified putative duplicate accessions to determine if they can be bulked. Using ten microsatellite primer pairs, we genotyped two bulks of B. rapa L. ssp. dichotoma (Roxb.) Hanelt comprising four accessions and three bulks of B. rapa L. ssp. trilocularis (Roxb.) Hanelt comprising fourteen accessions, as well as four pairs of putatively duplicate accessions of B.␣napus L. Assignment tests on ten individual plants per accession were conducted using a model-based clustering method to arrive at probabilities of likelihood of accession assignment. The assignment tests indicated that one of the two bulks of B. rapa ssp. dichotoma involves genetically heterogeneous accessions. It was observed in the B. rapa ssp. trilocularis bulks that the component accessions could be differentiated into groups, with misassignments observed most frequent within groups. In B. napus, only one of the four pairs of putative duplicates showed significant genetic differentiation. The other three pairs of putative duplicates lack differences and support the creation of bulks. The results of the assignment tests were in agreement with cluster analyses and tests of population differentiation. Implications of these results in terms of germplasm management include the maintenance and/or re-creation of some Brassica germplasm bulks by excluding those accessions identified as being unique in this study.     

Improving ovary and embryo culture techniques for efficient resynthesis of <Emphasis Type="Italic">Brassica napus</Emphasis> from reciprocal crosses between yellow-seeded diploids <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

The primary aim of this study was to optimize in vitro culture protocols to establish an efficient reproducible culture system for different Brassica interspecific crosses, and to synthesize yellow-seeded Brassica napus (AACC) for breeding and genetical studies. Reciprocal crosses were carried out between three B. rapa L. ssp. oleifera varieties (AA) and five accessions of B. oleracea var. acephala (CC). All the parental lines were yellow-seeded except one accession of B. oleracea. Hybrids were obtained through either ovary culture from crosses B. rapa × B. oleracea, or embryo culture from crosses B. oleracea × B. rapa. A higher rate of hybrid production was recorded when ovaries were cultured at 4–7 days after pollination (DAP). Of different culture media, medium E (MS with half strength macronutrients) showed good response for ovaries from all the crosses, the highest rate of hybrid production reaching 45% in B. rapa (1151) × B. oleracea (T₂). In embryo culture, the hybrid rate was significantly enhanced at 16–18 DAP, up to 48.1% in B. oleracea (T₃) × B. rapa (JB₂). The combinations of optimal DAP for excision and media components increased recovery of hybrids for ovary and embryo culture, and constituted an improved technique for B. rapa × B. oleracea crosses. In addition, yellow seeds were obtained from progenies of two crosses, indicating the feasibility of developing yellow-seeded B. napus through the hybridization between yellow-seeded diploids B. rapa and B. oleracea var. acephala.