Molecular Characterization of Lactobacillus plantarum Genes for β-Ketoacyl-Acyl Carrier Protein Synthase III (fabH) and Acetyl Coenzyme A Carboxylase (accBCDA), Which Are Essential for Fatty Acid Biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded β-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the β and α subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.     

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Analysis of the only plastid genome-encoded fatty acid biosynthesis gene reveals functions in plastid division and seed development, and antagonistic roles in autotrophic and heterotrophic growth.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Community Structure and Function of Planktonic Crenarchaeota: Changes with Depth in the South China Sea

Marine Crenarchaeota represent a widespread and abundant microbial group in marine ecosystems. Here, we investigated the abundance, diversity, and distribution of planktonic Crenarchaeota in the epi-, meso-, and bathypelagic zones at three stations in the South China Sea (SCS) by analysis of crenarchaeal 16S rRNA gene, ammonia monooxygenase gene amoA involved in ammonia oxidation, and biotin carboxylase gene accA putatively involved in archaeal CO₂ fixation. Quantitative PCR analyses indicated that crenarchaeal amoA and accA gene abundances varied similarly with archaeal and crenarchaeal 16S rRNA gene abundances at all stations, except that crenarchaeal accA genes were almost absent in the epipelagic zone. Ratios of the crenarchaeal amoA gene to 16S rRNA gene abundances decreased ~2.6 times from the epi- to bathypelagic zones, whereas the ratios of crenarchaeal accA gene to marine group I crenarchaeal 16S rRNA gene or to crenarchaeal amoA gene abundances increased with depth, suggesting that the metabolism of Crenarchaeota may change from the epi- to meso- or bathypelagic zones. Denaturing gradient gel electrophoresis profiling of the 16S rRNA genes revealed depth partitioning in archaeal community structures. Clone libraries of crenarchaeal amoA and accA genes showed two clusters: the “shallow” cluster was exclusively derived from epipelagic water and the “deep” cluster was from meso- and/or bathypelagic waters, suggesting that niche partitioning may take place between the shallow and deep marine Crenarchaeota. Overall, our results show strong depth partitioning of crenarchaeal populations in the SCS and suggest a shift in their community structure and ecological function with increasing depth.     

Seed-specific,developmentally regulated genes of peanut

Four cDNAs of seed-specific and developmentally regulated peanut (Arachis hypogaea L.) genes were identified by differential screening of a peanut-seed cDNA library using cDNA probes constructed from mRNAs isolated from immature and mature stages of the seed. Northern analysis, probed with the four cloned cDNAs, indicated that the genes represented by two cDNAs were expressed abundantly early in seed development, while another two were abundantly expressed later at the cell-expansion stages of seed development. These four genes did not show expression in roots, pegs or leaves. However, one of the early expressed genes was seed coat-specific. One of the clones, Psc11, had significant sequence similarity to subtilisin-like genes in Arabidopsis and soybean. Clones Psc32 and Psc33 had significant similarity to the peanut allergen genes Ara h II and Ara h 6, respectively. The sequence of clone Psc12 was unique and did not show significant similarity to any sequence in the databases. One of the four seed-specific clones showed restriction fragment length polymorphism (RFLP) among peanut lines representing the four peanut botanical varieties. These findings indicate that polymorphism exists in peanut seed-storage genes. This contrasts with other genes previously used for genetic mapping of cultivated peanut. Received: 1 September 2000 / Accepted: 4 May 2001     

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

The stearoyl–acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven single-nucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.     

Acetyl-CoA Carboxylase in Higher Plants: Most Plants Other Than Gramineae Have Both the Prokaryotic and the Eukaryotic Forms of This Enzyme

The presence and the absence of a prokaryote type and a eukaryotetype of acetyl-CoA carboxylase (EC 6.4.1.2 [EC] ; ACCase) were examinedin members of 28 plant families by two distinct methods: thedetection of biotinylated subunits of ACCase with a streptavidinprobe, and the detection of the accD gene, which encodes a subunitof the prokaryotic ACCase, by Southern hybridization analysis.The protein extracts of all the plants studied contained a biotinylatedpolypeptide of 220 kDa, which was probably the eukaryotic ACCase.All the plants but those belonging to Gramineae also containeda biotinylated polypeptide of ca. 35 kDa, which is a putativesubunit of the prokaryotic ACCase. In all plants but those inGramineae, the ca. 35 kDa polypeptide was found in the proteinextracts of plastids, while the 220 kDa polypeptide was absentfrom these plastid extracts. The plastid extracts of the plantsin Gramineae contained the 220 kDa polypeptide, as did the homogenatesof the leaves. Southern hybridization analysis demonstratedthat all the plants but those in the Gramineae contained theaccD gene. These findings suggest that most higher plants havethe prokaryotic ACCase in the plastids and the eukaryotic ACCasein the cytosol. Only Gramineae plants might contain the eukaryoticACCases both in the plastids and in the cytosol. The originof the plastid-located eukaryotic ACCase in Gramineae is discussedas the first possible example of substitution of a plastid geneby a nuclear gene for a non-ribosomal component. ⁴Present address: Plant-Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako,351-01 Japan ⁵Present address: Laboratory of Plant Molecular Biology, Schoolof Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya,464-01 Japan     

Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China

It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO₂ using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66–96 °C) and pH (4.3–9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO₂ fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.     

The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.     

Role of an essential acyl coenzyme A carboxylase in the primary and secondary metabolism of Streptomyces coelicolor A3(2)

Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109-3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites.     

Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses

We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution. Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them. These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK. The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods. Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons. On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA. On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA. The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics.     

Dehydrin genes and their expression in recalcitrant oak (<Emphasis Type="Italic">Quercus robur</Emphasis>) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for Y_nSK_n type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative K_n type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.     

Loss of the Acetyl-CoA Carboxylase (accD) Gene in Poales

The loss of a gene is a rare genome-shaping event and as such, contributes important information to our understanding of phylogenetic relationships between genes and between species. Deletion of a gene can help to define a lineage. Here, we utilize the deletion of the chloroplast gene encoding the acetyl-CoA carboxylase subunit D (accD) to help us define lineages based on its presence or absence in monocot plants specifically in Poales. Southern blots were constructed and probed for the presence of the accD gene. The existence of the portion of the accD gene represented by the probe was also verified by PCR and sequencing. Sequences were utilized for assembly of gene trees to link the absence or partial loss of the gene with a particular lineage. Here, we report new information adding accD gene presence in the Xyridaceae, pseudogene presence in the Flagellariaceae, and the absence of accD in Restionaceae and Joinvilleaceae. Based on our findings and the available data for accD sequences in Poales, we propose a model for accD loss beginning with a single event creating a pseudogene in the common ancestor to the restiid and graminid clades within Poales. This model also suggests that this pseudogene is carried as the ancestral state throughout most of the divergence of the Poales, a condition that would explain the highly varied pattern of accD pseudogene presence or gene absence in members of the restiid and graminid clades.     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

Characterization of Five Putative Acyl Carrier Protein (ACP) Isoforms from Developing Seeds of <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.

Five putative acyl carrier protein (ACP) cDNAs were isolated from developing peanut seeds by searching ESTs of a peanut immature-seed cDNA library and PCR-based cloning. Five peanut ACPs contained a strictly conserved Ser residue in the Asp-Ser-Leu (DSL) motif, which is an important characteristic of ACPs in plants and bacteria. Three AhACPs, AhACP1, AhACP4, AhACP5, were predicted to be located in chloroplast, while two AhACPs, AhACP2 and AhACP3, in mitochondria. Comparison of genomic DNA and cDNA sequences demonstrated three chloroplast ACPs (cpACPs) comprising of four exons and three introns while two mitochondrial ACPs (mtACPs) contained two exons and one intron. More than two homologs of each AhACP were expressed in developing peanut seeds. Most homologs were verified by corresponding genomic DNA sequences. Semi-quantitative RT-PCR analysis suggested AhACP1 was a seed-predominant ACP isoform. AhACP4 and AhACP5 showed same mRNA profile in different organs and during seed development. Two mtACPs expressed highly in peanut flower tissue which was distinct from three cpACPs.     

Dynamic Histone Acetylation of Late Embryonic Genes during Seed Germination

Histone acetylation is involved in the regulation of gene expression in plants and eukaryotes. Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from histones, which is associated with the repression of gene expression. To study the role of histone acetylation in the regulation of gene expression during seed germination, trichostatin A (TSA), a specific inhibitor of histone deacetylase, was used to treat imbibing Arabidopsis thaliana seeds. GeneChip arrays were used to show that TSA induces up-regulation of 45 genes and down-regulation of 27 genes during seed germination. Eight TSA-up-regulated genes were selected for further analysis – RAB18, RD29B, ATEM1, HSP70 and four late embryogenesis abundant protein genes (LEA). A gene expression time course shows that these eight genes are expressed at high levels in the dry seed and repressed upon seed imbibition at an exponential rate. In the presence of TSA, the onset of repression of the eight genes is not affected but the final level of repressed expression is elevated. Chromatin immunoprecipitation and HDAC assays show that there is a transient histone deacetylation event during seed germination at 1 day after imbibition, which serves as a key developmental signal that affects the repression of the eight genes. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.     

Inheritance of seed coat color genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) and tagging the genes using SRAP,SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F₁, F₂, F₃ and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F₂ and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.