Quantitative trait loci (QTLs) contributing to yellow seed colour and acid detergent fibre (ADF) were localized and compared in 3 mapping populations developed from 2 crosses (designated 'YE1' and 'YE2') between 2 distinct sources of true-breeding yellow-seeded oilseed rape (Brassica napus) and 2 different black-seeded genotypes. A clear correlation was observed between seed colour and ADF content in both crosses. In all 3 populations, a major QTL, with a large effect on both seed colour and ADF in multiple environments, was detected at the same position on chromosome N18. In YE1, a second minor QTL, with a small effect on seed colour but not on ADF content, was localized on chromosome N1. In YE2, no QTL was observed on N1; however, 2 minor seed-colour loci were localized to N15 and N5. A second major QTL for ADF was localized in YE1 on N13; in YE2, no other QTLs for ADF were detected. Combined QTL and segregation data for seed colour and ADF content in the different populations suggest that a partially dominant B. napus gene for seed colour on N18 contributes to a reduction in fibre content in different yellow-seeded B. napus genotypes. The other QTLs that were identified appear to represent different genes in the 2 yellow-seeded rapeseed sources, which, in each case, affect only fibre content or seed colour, respectively. Potential candidate genes and implications for marker-assisted breeding of oilseed rape with reduced seed dietary fibre content are discussed.

Colocalization of a partially dominant gene for yellow seed colour with a major QTL influencing acid detergent fibre (ADF) content in different crosses of oilseed rape (Brassica napus)

Molecular dissection of the Brassica yellow seed trait has been the subject of intense investigation. Arabidopsis thaliana TRANSPARENT TESTA 12 (AtTT12) encodes a multidrug and toxic compound extrusion (MATE) transporter involved in seed coat pigmentation. Two, one, and one full-length TT12 genes were isolated from B. napus, B. oleracea, and B. rapa, respectively, and Southern hybridization confirmed these gene numbers, implying loss of some of the triplicated TT12 genes in Brassica. BnTT12-1, BnTT12-2, BoTT12, and BrTT12 are 2,714, 3,062, 4,760, and 2,716 bp, with the longest mRNAs of 1,749, 1,711, 1,739, and 1,752 bp, respectively. All genes contained alternative transcriptional start and polyadenylation sites. BrTT12 and BoTT12 are the progenitors of BnTT12-1 and BnTT12-2, respectively, validating B. napus as an amphidiploid. All Brassica TT12 proteins displayed high levels of identity (>99%) to each other and to AtTT12 (>92%). Brassica TT12 genes resembled AtTT12 in such basic features as MatE/NorM CDs, subcellular localization, transmembrane helices, and phosphorylation sites. Plant TT12 orthologs differ from other MATE proteins by two specific motifs. Like AtTT12, all Brassica TT12 genes are most highly expressed in developing seeds. However, a range of organ specificity was observed with BnTT12 genes being less organ-specific. TT12 expression is absent in B. rapa yellow-seeded line 06K124, but not downregulated in B. oleracea yellow-seeded line 06K165. In B. napus yellow-seeded line L2, BnTT12-2 expression is absent, whereas BnTT12-1 is expressed normally. Among Brassica species, TT12 genes are differentially related to the yellow seed trait. The molecular basis for the yellow seed trait, in Brassica, and the theoretical and practical implications of the highly variable intron 1 of these TT12 genes are discussed.

TRANSPARENT TESTA 12 genes from Brassica napus and parental species: cloning, evolution, and differential involvement in yellow seed trait.

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F2, BC1 and F1-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population-derived from the cross Hui5148-2xNo.2127-17-of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.).

Cinnamate 4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which synthesizes numerous secondary metabolites to participate in development and adaption. Two C4H isoforms, the 2192-bp BnC4H-1 and 2108-bp BnC4H-2, were cloned from oilseed rape (Brassica napus). They both have two introns and a 1518-bp open reading frame encoding a 505-amino-acid polypeptide. BnC4H-1 is 57.73 kDa with an isoelectric point of 9.11, while 57.75 kDa and 9.13 for BnC4H-2. They share only 80.6% identities on nucleotide level but 96.6% identities and 98.4% positives on protein level. Showing highest homologies to Arabidopsis thaliana C4H, they possess a conserved p450 domain and all P450-featured motifs, and are identical to typical C4Hs at substrate-recognition sites and active site residues. They are most probably associated with endoplasmic reticulum by one or both of the N- and C-terminal transmembrane helices. Phosphorylation may be a necessary post-translational modification. Their secondary structures are dominated by alpha helices and random coils. Most helices locate in the central region, while extended strands mainly distribute before and after this region. Southern blot indicated about 9 or more C4H paralogs in B. napus. In hypocotyl, cotyledon, stem, flower, bud, young- and middle-stage seed, they are co-dominantly expressed. In root and old seed, BnC4H-2 is dominant over BnC4H-1, with a reverse trend in leaf and pericarp. Paralogous C4H numbers in Brassicaceae genomes and possible roles of conserved motifs in 5' UTR and the 2nd intron are discussed.

/pdf/molecular-cloning-of-two-genes-encoding-cinnamate-4-56aou4o71o.pdf

Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus).

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.

Improving ovary and embryo culture techniques for efficient resynthesis of Brassica napus from reciprocal crosses between yellow-seeded diploids B. rapa and B. oleracea

Most oilseed rape, Brassica napus, cultivars are black-seeded. The progenitor species, Brassica rapa, has either yellow or black seeds, while known cultivars of the other progenitor species Brassica oleracea/alboglabra have black seeds. To determine which chromosomes of B. alboglabra are carriers of seed colour genes, B. rapa-alboglabra monosomic addition lines were produced from a B. napus resynthesized from yellow-seeded B. rapa and brown/black-seeded B. alboglabra. Eight out of nine possible lines have been developed and transmission frequencies of the alien chromosomes were estimated. Three B. alboglabra chromosomes in three of these lines influenced seed colour. B. rapa plants carrying alien chromosome 1 exhibited a maternal control of seed colour and produced only brown seeds, which gave rise to plants with either yellow or brown seeds. However, B. rapa plants carrying alien chromosome 4 or another as yet unidentified alien chromosome exhibited an embryonal control of seed colour and produced a mixture of yellow and brown seeds. The yellow seeds gave rise to yellow-seeded plants, while the brown seeds gave rise to plants that yielded a mixture of yellow and brown seeds, depending on the absence or presence, respectively, of the B. alboglabra chromosome. Consequently, both maternal and embryonal control of seed colour are expected to contribute to the black-seeded phenotype of oilseed rape.

Maternal and embryonal control of seed colour by different Brassica alboglabra chromosomes

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Maternal and embryonal control of seed colour by different Brassica alboglabra chromosomes