Molecular Characterization, Gene Evolution, and Expression Analysis of the Fructose-1, 6-bisphosphate Aldolase (FBA) Gene Family in Wheat (Triticum aestivum L.).

Fructose-1, 6-bisphosphate aldolase (FBA) is a key plant enzyme that is involved in glycolysis, gluconeogenesis, and the Calvin cycle. It plays significant roles in biotic and abiotic stress responses, as well as in regulating growth and development processes. In the present paper, 21 genes encoding TaFBA isoenzymes were identified, characterized, and categorized into three groups: class I chloroplast/plastid FBA (CpFBA), class I cytosol FBA (cFBA), and class II chloroplast/plastid FBA. By using a prediction online database and genomic PCR analysis of Chinese Spring nulli-tetrasomic lines, we have confirmed the chromosomal location of these genes in 12 chromosomes of four homologous groups. Sequence and genomic structure analysis revealed the high identity of the allelic TaFBA genes and the origin of different TaFBA genes. Numerous putative environment stimulus-responsive cis-elements have been identified in 1,500-bp regions of TaFBA gene promoters, of which the most abundant are the light-regulated elements (LREs). Phylogenetic reconstruction using the deduced protein sequence of 245 FBA genes indicated an independent evolutionary pathway for the class I and class II groups. Although, earlier studies have indicated that class II FBA only occurs in prokaryote and fungi, our results have demonstrated that a few class II CpFBAs exist in wheat and other closely related species. Class I TaFBA was predicted to be tetramers and class II to be dimers. Gene expression analysis based on microarray and transcriptome databases suggested the distinct role of TaFBAs in different tissues and developmental stages. The TaFBA 4-9 genes were highly expressed in leaves and might play important roles in wheat development. The differential expression patterns of the TaFBA genes in light/dark and a few abiotic stress conditions were also analyzed. The results suggested that LRE cis-elements of TaFBA gene promoters were not directly related to light responses. Most TaFBA genes had higher expression levels in the roots than in the shoots when under various stresses. Class I cytosol TaFBA genes, particularly TaFBA10/12/18 and TaFBA13/16, and three class II TaFBA genes are involved in responses to various abiotic stresses. Class I CpFBA genes in wheat are apparently sensitive to different stress conditions.

Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE5.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-related protein kinase3-type protein kinase, SOS2-like protein kinase5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, abscisic acid-insensitive5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-like calcium binding proteins) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).

BolOST1, an ortholog of Open Stomata 1 with alternative splicing products in Brassica oleracea, positively modulates drought responses in plants.

Open Stomata 1 (OST1), an ABA-activated sucrose non-fermenting 1 (SNF1)-related protein kinase, is critical for plant drought responses. We investigated the functions of two splicing isoforms of the OST1 ortholog in Brassica oleracea (BolOST1). BolOST1 expression was found to be dramatically induced by drought and high-salt stress, and the ectopic expression of BolOST1 restored the drought-sensitive phenotype of ost1. Subcellular localization revealed that BolOST1 is localized in both the nucleus and cytoplasm. BolOST1 was also demonstrated to phosphorylate the N-terminal fragment of ABI5 (ABA Insensitive 5, ABI5-N). A firefly luciferase complementation assay revealed that BolOST1 interacts with both BolABI5 and an ABI1 ortholog in B. oleracea (BolABI1). Overall, these results suggest that BolOST1 is a functional SnRK2-type protein kinase and that the early ABA signaling network may be conserved between Arabidopsis and cabbage.

Negative regulation of abscisic acid signaling by the Brassica oleracea ABI1 ortholog.

ABI1 (ABA Insensitive 1) is an important component of the core regulatory network in early ABA (Abscisic acid) signaling. Here, we investigated the functions of an ABI1 ortholog in Brassica oleracea (BolABI1). The expression of BolABI1 was dramatically induced by drought, and constitutive expression of BolABI1 confers ABA insensitivity upon the wild-type. Subcellular localization and phosphatase assays reveal that BolABI1 is predominantly localized in the nucleus and harbors phosphatase activity. Furthermore, BolABI1 interacts with a homolog of OST1 (OPEN STOMATA 1) in B. oleracea (BolOST1) and can dephosphorylate ABI5 (ABA Insensitive 5) in vitro. Overall, these results suggest that BolABI1 is a functional PP2C-type protein phosphatase that is involved in the negative modulation of the ABA signaling pathway.

Molecular characterization of an ABA insensitive 5 orthologue in Brassica oleracea.

ABI5 (ABA insensitive 5), a bZIP (Basic leucine zipper) transcription factor, has been shown to be a major mediator of plant ABA responses during seed germination. Although the molecular basis of ABI5-modulated processes has been well demonstrated in Arabidopsis thaliana, its identity and function in cabbage (Brassica oleracea var. capitata L.) remain elusive. Here, we describe our identification of BolABI5 (an ABI5 orthologue in B.oleracea) as a functional bZIP transcription factor in the modulation of plant ABA responses. Expression of BolABI5 was dramatically induced by drought stress and exogenous ABA. Heterogeneous expression of BolABI5 rescued the insensitive phenotype of Arabidopsis abi5-1 to ABA during seed germination. Subcellular localization and trans-activation assays revealed that BolABI5 was localized in the nucleus and possessed DNA binding and trans-activation activities. Deletion of the bZIP domain generated BolABI5ΔbZIP, which no longer localized exclusively in the nucleus and had almost no detectable DNA-binding or trans-activation activities. Overall, these results suggest that BolABI5 may function as ABI5 in the positive regulation of plant ABA responses.

Effect of low temperature on chlorophyll biosynthesis in albinism line of wheat (Triticum aestivum) FA85.

The 'stage albinism line of winter wheat' FA85 exhibits a severe block in chlorophyll (Chl) biosynthesis with prolonged low-temperature treatment. The correlations between leaf color and low temperature provide more comprehensive understanding of low temperature as an environmental signal that regulate the metabolic changes in the entire Chl-synthesizing pathway. In this study, we investigated differences in Chl biosynthesis between leaves of Aibian1 and FA85 by measuring their Chl precursors and heme content, transcripts for key genes of Chl biosynthesis and key enzyme activities. With prolonged low-temperature treatment, the Chl content gradually decreased, but Chl precursors, including protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide (Pchlide), simultaneously accumulated. Parallel to the decline in Chl content, the protoporphyrin IX distribution toward Chl synthesis was less than that in heme synthesis in the leaves of FA85. Corresponding to the change of protoporphyrin IX distribution, the relative changes in magnesium chelatase (EC 6.6.1.1) and ferrochelatase (EC 4.99.1.1) activities in the leaves of FA85 also indirectly reflected channeling of the metabolic flow into heme rather than Chl. A drastic loss in the transcripts for Pchlide oxidoreductase (EC 1.3.1.33) and Chl synthase (EC 2.5.1.62) accounted for a decrease in the metabolic flux and the re-direction of metabolites. The high-level accumulations of Chl precursors and traces of Chl in the leaves of FA85 suggest that a severe block between the steps from Pchlide to Chl formation during Chl biosynthesis is partially derived from the transcriptional downregulation of Pchlide oxidoreductase and Chl synthase.

Functional analysis of the two Brassica AP3 genes involved in apetalous and stamen carpelloid phenotypes.

The Arabidopsis homeotic genes APETALA3 (AP3) and PISTILLATA (PI) are B genes which encode MADS-box transcription factors and specify petal and stamen identities. In the current study, the stamen carpelloid (SC) mutants, HGMS and AMS, of B. rapa and B. napus were investigated and two types of AP3 genes, B.AP3.a and B.AP3.b, were functional characterized. B.AP3.a and B.AP3.b share high similarity in amino acid sequences except for 8 residues difference located at the C-terminus. Loss of this 8 residues in B.AP3.b led to the change of PI-derived motifs. Meanwhile, B.AP3.a specified petal and stamen development, whereas B.AP3.b only specified stamen development. In B. rapa, the mutations of both genes generated the SC mutant HGMS. In B. napus that contained two B.AP3.a and two B.AP3.b, loss of the two B.AP3.a functions was the key reason for the apetalous mutation, however, the loss-of-function in all four AP3 was related to the SC mutant AMS. We inferred that the 8 residues or the PI-derived motif in AP3 gene probably relates to petal formation.

Fine mapping of a male sterility gene MS-cd1 in Brassica oleracea.

A dominant male sterility (DGMS) line 79-399-3, developed from a spontaneous mutation in Brassica oleracea var. capitata, has been widely used in production of hybrid cultivars in China. In this line, male sterility is controlled by a dominant gene Ms-cd1. In the present study, fine mapping of Ms-cd1 was conducted by screening a segregating population Ms79-07 with 2,028 individuals developed by four times backcrossing using a male sterile Brassica oleracea var. italica line harboring Ms-cd1 as donor and Brassica oleracea var. alboglabra as the recipient. Bulked segregation analysis (BSA) was performed for the BC(4) population Ms79-07 using 26,417 SRAP primer SRAPs and 1,300 SSRs regarding of male sterility and fertility. A high-resolution map surrounding Ms-cd1 was constructed with 14 SRAPs and one SSR. The SSR marker 8C0909 was closely linked to the MS-cd1 gene with a distance of 2.06 cM. Fourteen SRAPs closely linked to the target gene were identified; the closest ones on each side were 0.18 cM and 2.16 cM from Ms-cd1. Three of these SRAPs were successfully converted to dominant SCAR markers with a distance to the Ms-cd1 gene of 0.18, 0.39 and 4.23 cM, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region about 600 kb in scaffold 000010 on chromosomeA10 in B. rapa and on chromosome 5 in A. thaliana. These results provide additional information for map-based cloning of the Ms-cd1 gene and will be helpful for marker-assisted selection (MAS).

[Prokaryotic expression, purification and preparation of polyclonal antibody for wheat grain peroxidase WP1 gene].

Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.

Targets for human encoded microRNAs in HBV genes.

MicroRNAs (miRNAs) are increasingly being shown to play vital roles in development, apoptosis, and oncogenesis by interfering with gene expression at the post-transcriptional level. miRNAs, in principle, can contribute to the repertoire of host-pathogen interactions during infection by the Hepatitis B virus (HBV). Using a consensus-scoring approach, high-scoring miRNA-target pairs were selected, which were identified by four well-established target-prediction softwares. The miRNAs miR-7, miR196b, miR433, and miR511 target the polymerase or S gene of HBV, miR205 targets the X gene, and miR345 targets the preC gene. The minimum free-energy values for the bound complexes were the lowest, and the rules so far observed for miRNA-target pairing, namely, (1) pairing at a continuous stretch of 6-7 bases toward the 5'-end of the miRNA and (2) incomplete complementarity with the target sequence, were found to be valid. The target regions were highly conserved across the various clades of HBV. miRNA expression profiles from previously reported Solexa-sequencing based experiments showed that the four human miRNAs are expressed in the liver. This is the first report of human miRNAs that can target crucial HBV genes.

Verotoxin 2 enhances adherence of enterohemorrhagic Escherichia coli O157:H7 to intestinal epithelial cells and expression of {beta}1-integrin by IPEC-J2 cells.

Verotoxin (VT) has been implicated in the promotion of adherence to and colonization of intestinal epithelial cells by enterohemorrhagic Escherichia coli (EHEC) O157:H7. The present study investigated the effect of VT2 on the adherence of EHEC O157:H7 strain 86-24 to porcine jejunal (IPEC-J2), human colon (CaCo-2), and human laryngeal carcinoma (HEp-2) cell lines and on the expression in IPEC-J2 cells of synthases for beta1-integrin and nucleolin, both of which are implicated in bacterial adherence. The effect on expression of globotriaosylceramide (Gb3) synthase, the receptor for VT, was also examined. Data were obtained by adherence assays and quantitative reverse transcriptase PCR, using EHEC O157 strain 86-24, a vt2 deletion mutant, a vt2 phage-negative strain, and complemented mutants in which the vt2 gene was restored. Compared with the adherence of the parent and complemented mutant strains, the vt2-negative strains adhered significantly less to all three types of cells. Adherence of the wild-type EHEC strain to IPEC-J2 cells was accompanied by increased expression of beta1-integrin, nucleolin, and Gb3 synthase. IPEC-J2 cells in association with wild-type EHEC O157:H7 or the complemented mutants expressed higher levels of beta1-integrin than did cells in association with the vt2-negative strains or with no bacteria. Expression of nucleolin was decreased by association with the vt2-negative mutant, but complementation failed to restore wild-type expression. The data indicate that VT2 plays a role in the adherence of EHEC O157:H7 to intestinal epithelial cells, possibly by increasing the expression of the host receptor beta1-integrin.

Cloning and identification of a novel P-II class snake venom metalloproteinase from Gloydius halys.

Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation-inhibition ability.

Proteome analysis of chloroplast proteins in stage albinism line of winter wheat (triticum aestivum) FA85.

The "stage albinism line of winter wheat" FA85 was a specific natural mutant strain on leaf color. This physiological mutation was controlled by cytogene. In order to reveal the genetic and biochemical mechanism of albinism, 2-DE was used to investigate the difference of chloroplast protein expression pattern between FA85 and its parent wheat Aibian 1. From the results of 2-DE gels analysis, approximately 683 spots were detected on each gel, and 57 spots were expressed differently at least two-fold. Using MALDI-TOF/TOF MS, 14 of 57 spots were identified, which could be categorized into four classes: carbon metabolism, energy metabolism, defense/stress response and signal transduction. Compared with the parent wheat, the expression of ATPase-gamma and GP1-alpha was up-regulated in FA85, and of other proteins was down-regulated. Together, we concluded that the expression of chloroplast proteins had changed obviously in FA85, which might be related to the leaf color mutant.

[Prokaryotic expression, purification and preparation of polyclonal antibody for human SUMO-2 gene].

AIM: To construct a prokaryotic expression vector of human SUMO-2, purify GST-SUMO2-SUMO2 fusion protein produced by the expression system, and prepare its antiserum. METHODS: The human SUMO-2 gene was amplified by PCR. The target fragment digested by the enzyme was cloned into a pET41a(+) expression vector and then transfected into E.coli. BL21 (DE3) pLysS, in which GST-SUMO2-SUMO2 fusion protein was induced by IPTG. After the soluble protein was purified by GST affinity chromatography and by identified by SDS-PAGE, the rabbits were immunized with the fusion protein and the antiserum was obtained. RESULTS: DNA sequence analysis showed the cloned SUMO-2 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blot showed that the GST-SUMO2-SUMO2 fusion protein was about 52 kDa, which was mainly the soluble protein of E.coli and could be purified by GST affinity chromatography. The result of ELISA was positive and Western blot confirmed the antiserum reacted specifically to SUMO-2 protein. CONCLUSION: SUMO-2 protein and its specific polyclonal antibody have been prepared, which provides a basis for the establishment of immunoassays of human SUMO-2.

Identification and verification of microRNA in wheat (Triticum aestivum).

MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in both plants and animals. A large number of miRNAs has been identified from various animals and model plant species such as Arabidopsis thaliana and rice (Oryza sativa); however, characteristics of wheat (Triticum aestivum) miRNAs are poorly understood. Here, computational identification of miRNAs from wheat EST sequences was preformed by using the in-house program GenomicSVM, a prediction model for miRNAs. This study resulted in the discovery of 79 miRNA candidates. Nine out of 22 miRNA representatives randomly selected from the 79 candidates were experimentally validated with Northern blotting, indicating that prediction accuracy is about 40%. For the 9 validated miRNAs, 59 wheat ESTs were predicted as their putative targets.

Expression, purification, and activity identification of Alfimeprase in Pichia pastoris.

Alfimeprase (ALF) is a truncated form of non-hemorrhagic zinc metalloproteinase fibrolase. In order to achieve a high level secretion and full activity expression of ALF, the Pichia pastoris (P. pastoris) expression system was used. ALF coding sequence fused with a 6 *histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector pPIC9K and then expressed in P. pastoris strains of GS115 and KM71 by methanol induction. SDS-PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to mouse anti-His. tag monoclonal antibody. Under the optimized culture parameters of pH value, initial A(600) value, methanol daily addition concentration and induction time length, the production of rALF reached up to 510 mg/L and 465 mg/L of the GS115 and KM71 transformants, respectively. It also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purity of rALF was as high as 96%. The fibrinolytic activity of rALF revealed by the modified fibrin plate method indicated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. The improved expression system will facilitate further studies and industrial production of ALF.

Identification of promoters of two dehydrogenase genes in Ketogulonicigenium vulgare DSM 4025 and their strength comparison in K. vulgare and Escherichia coli.

Promoters of sorbose dehydrogenase gene sdh and sorbosone dehydrogenase gene sndh (Psdh and Psndh) in Ketogulonicigenium vulgare DSM 4025 were identified. The transcription initiation site (TIS) of Psdh was guanine 74 bp upstream of the start codon of sdh and the TIS of Psndh was adenine 113 bp upstream of the first codon of sndh. Comparing Psdh and Psndh, consensus sequences were found, which were TAVCVT (V=A, C or G) and THGAHC (H=A, C or T) for their putative -10 and -35 regions, respectively, and the spans between the 2 regions were 17 bp. Psdh and Psndh promoters may be constitutive in K. vulgare DSM 4025 when cultured in HJ medium. Semiquantitative RT-PCR analysis showed that the Psdh promoter was about 2.5 times stronger than Psndh in strength in K. vulgare DSM 4025. In Escherichia coli, Psdh and Psndh demonstrated strong activity with the former about two times stronger than the latter. DCIP decoloration method and reporter plasmids pSDH or pSNDH may be applied to discover promoters of genes in E. coli and to determine their strength in one step.

HBV-encoded microRNA candidate and its target.

MicroRNAs (miRNAs) are a group of short (approximately 22 nt) noncoding RNAs that specifically regulate cellular gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of approximately 70 nt, are processed into mature miRNAs by cellular RNases III. To date, hundreds of miRNAs and their corresponding targets have been reported in kinds of species. Although only a few of these miRNA/target pairs have been functionally verified, some do play important roles in regulating normal development and physiology. Several viruses (e.g. the Epstein-Barr virus and human herpesvirus Kaposi's sarcoma-associated herpesvirus) has been reported to encode miRNAs. Here, we extend the analysis of miRNA-encoding potential to the Hepatitis B virus (HBV). Using computational approaches, we found that HBV putatively encodes only one candidate pre-miRNA. We then matched deduced mature miRNA sequence from this pre-miRNA against a database of 3' untranslated sequences (UTR) from the human genome. Surprisingly, none of cellular transcripts could potentially be targeted by the viral miRNA (vmiRNA) sequence. However, one viral mRNA was found to be targeted by the vmiRNA when we searched the target from viral mRNAs. We propose that HBV has evolved to use vmiRNAs as a means to regulate its own gene expression for its benefit.

Cytological and molecular characterization of a novel monogenic dominant GMS in Brassica napus L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5'-CTATGCCGAC-3') gave a repeatable 1500-bp DNA polymorphic segment S243(1500) between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243(1500) is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.