Male Sterility In Chinese Cabbage Research Articles

Transcriptome analysis reveals the potential lncRNA-mRNA modules involved in genetic male sterility and fertility of Chinese cabbage (brassica rapa L. ssp. pekinensis).

Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. In this study, we characterized a recessive genic male sterile mutant (366-2S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2S) and the wild-type male fertile line (366-2F). We identified 385 differentially expressed lncRNAs between the 366-2F and 366-2S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2S and 366-2F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3C (β-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3C (pectinesterase) and MSTRG.13,532-BraA05g030320.3C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

Mutations in BrABCG26, encoding an ATP-binding cassette transporter, are responsible for male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The gene BrABCG26 responsible for male sterility of Chinese cabbage was confirmed by two allelic mutants. Male-sterile lines are an important way of heterosis utilization in Chinese cabbage. In this study, two allelic male-sterile mutants msm3-1 and msm3-2 were obtained from a Chinese cabbage double haploid (DH) line 'FT' by using EMS-mutagenesis. Compared to the wild-type 'FT,' the stamens of mutants were completely degenerated and had no pollen, and other characters had no obvious differences. Cytological observation revealed that the failure of vacuolation of the mononuclear microspore, accompanied by abnormal tapetal degradation, resulted in anther abortion in mutants. Genetic analysis showed thata recessive gene controlled the mutant trait. MutMap combined with kompetitive allele specific PCR genotyping analyses showed that BraA01g038270.3C, encoding a transporter ABCG26 that played a vital role in pollen wall formation, was the candidate gene for msm3-1, named BrABCG26. Compared with wild-type 'FT,' the mutations existed on the second exon (C to T) and the sixth exon (C to T) of BrABCG26 gene in mutants msm3-1 and msm3-2, leading to the loss-of-function truncated protein, which verified the BrABCG26 function in stamen development. Subcellular localization and expression pattern analysis indicated that BrABCG26 was localized in the nucleus and was expressed in all organs, with the highest expression in flower buds. Compared to the wild-type 'FT,' the expressions of BrABCG26 were significantly reduced in flower buds and anthers of mutants. Promoter activity analysis showed that a strong GUS signal was detected in flower buds. These results indicated that BrABCG26 is responsible for the male sterility of msm3 mutants in Chinese cabbage.

Mutation in BrGGL7 gene encoding a GDSL esterase / lipase causes male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

MutMap and KASP analyses revealed that the BrGGL7 gene is responsible for the male-sterile trait of ftms1 in Chinese cabbage, with functional verification in Arabidopsis. The application of a male-sterile line is an ideal approach of hybrid seed production in Chinese cabbage. In this study, we obtained a male-sterile mutant (ftms1) from the double haploid line 'FT' using ethyl methane sulfonate (EMS) mutagenesis. The mutant was completely sterile due to abnormal enlargement and vacuolization of the tapetum cells. A single recessive nuclear gene was found to control male sterility in the mutant, while MutMap and KASP analyses identified BraA05g022470.3C (BrGGL7), which encodes a GDSL esterase / lipase, as the candidate mutant gene. A single nucleotide substitution from C to T occurred within the domain of BrGGL7 in ftms1, resulting in premature translation termination in the fourth exon. Meanwhile, qRT-PCR analysis indicated that BrGGL7 was prominently expressed in the anthers, and expression was greater in the wild-type 'FT' than ftms1. Genetic complementation of the orthologous Arabidopsis ggl7 mutant further confirmed the role of BrGGL7 in pollen development. These findings suggest that BrGGL7 plays a fundamental role in pollen formation, providing important insight into the molecular mechanisms underlying male sterility in Chinese cabbage.

Investigation of the genes associated with a male sterility mutant (msm) in Chinese cabbage (Brassica campestris ssp. pekinensis) using RNA-Seq.

In Chinese cabbage, hybrid seed production is performed using male sterility lines, an important approach to heterosis utilization. In this study, a stably inherited male sterile mutant msm was obtained from the 'FT'-doubled haploid line of Chinese cabbage using isolated microspore culture combined with 60Co γ-ray mutagenesis. The genetic backgrounds of 'FT' and msm were highly consistent; however, compared with wild-type 'FT', msm exhibited completely degenerated stamens and no pollen phenotype. Other characters showed no significant differences. Cytological observations revealed that stamen abortion in msm begins during the tetrad period and that tapetum cells were abnormally expanded and highly vacuolated, leading to microspore abortion. Genetic analysis indicated that the msm mutant phenotype is controlled by a single recessive nuclear gene. Comparative transcriptome analysis of 'FT' and msm flower buds using RNA-Seq technology revealed 1653 differentially expressed genes, among which, a large number associated with male sterility were detected, including 64 pollen development- and pollen tube growth-related genes, 94 pollen wall development-related genes, 11 phytohormone-related genes, and 16 transcription factor-related genes. An overwhelming majority of these genes were down-regulated in msm compared with 'FT'. Furthermore, KEGG pathway analysis indicated that a variety of carbohydrate metabolic and lipid metabolic pathways were significantly enriched, which may be related to pollen abortion. The expression patterns of 24 male sterility-related genes were analyzed using qRT-PCR. In addition, 24,476 single-nucleotide polymorphisms and 413,073 insertion-deletion events were specifically detected in msm. These results will facilitate elucidation of the regulatory mechanisms underlying male sterility in Chinese cabbage.

ITRAQ-based proteomic analysis of fertile and sterile flower buds from a genetic male sterile line ‘AB01’ in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

To investigate the molecular basis of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis), we performed differential proteomic analysis using iTRAQ to identify differentially abundant proteins between fertile and sterile flower buds from the genetic male sterile line ‘AB01’. We identified 5932 high-confidence proteins; 1494 were differentially abundant between the two samples, including 749 up- and 745 down-regulated proteins. The up- and down-regulated proteins that could be essential for anther development and male sterility in sterile buds were mainly involved in (1) carbohydrate and energy metabolism (pyruvate dehydrogenase, glycolysis/gluconeogenesis, TCA cycle, starch and sucrose metabolism), (2) pollen wall synthesis and regulation (pectinesterase, polygalacturonase, pectate lyase, beta-galactosidase, glycosyl hydrolase), (3) protein synthesis and degradation (proteasome subunits, ribosome proteins, ABC transporters, RNA transport, protein processing in endoplasmic reticulum), (4) flavonoid biosynthesis, and (5) plant hormone signal transduction. We identified 10 genes/proteins that were both up-regulated and 122 that were both down-regulated in a conjoint analysis. Multiple reaction monitoring and qRT-PCR validation showed that the iTRAQ results were accurate and reliable. These findings will provide valuable information on proteins involved in anther development, and will contribute to the understanding of the molecular mechanism(s) that underlie male sterility in Chinese cabbage. Biological significanceChinese cabbage is an allogamous plant with bisexual flowers that displays significant heterosis. The application of male sterile lines is a very efficient way to produce hybrid seeds, which can generate stronger plants that develop more rapidly and produce higher yield. However, the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage is unknown. In this study, we used a quantitative proteomic approach (iTRAQ) to identify DAPs between fertile and sterile buds of the GMS line ‘AB01’. Subsequently, we also performed conjoined analysis of the iTRAQ results and our previously reported transcriptomics results. The aim of this research was to obtain the key DAPs and to identify the significantly enriched pathways involved in anther development and male sterility. These results may provide new insights into the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage.

Mapping of the male sterile mutant gene ftms in Brassica rapa L. ssp. pekinensis via BSR-Seq combined with whole-genome resequencing.

A male sterile mutant was created by 60Co γ-rays of microspores isolated from Chinese cabbage DH line 'FT'. A candidate gene for the male sterile trait was identified as Bra010198. Male sterility is used for hybrid seed production in Chinese cabbage. In this study, we derived a male sterile mutant (ftms) from Chinese cabbage DH line 'FT' by irradiating microspores with 60Co γ-rays and realized the rapid trait transformation from male fertility to sterility for creating valuable breeding materials. Genetic analysis indicated that the male sterile trait is controlled by a single recessive nuclear gene, ftms. Microspore development in mutant ftms was aborted at the tetrad stage and associated with severely retarded degeneration and vacuolation of tapetum. Using BSR-seq analysis, the candidate region for ftms was mapped on chromosome A05. A large F2 population was created, and the region was narrowed to approximately 1.7-Mb between markers Indel20 and Indel14 via linkage analysis. The recombination frequency was extremely suppressed because the region was located on the chromosome A05 centromere. Whole-genome resequencing of mutant ftms and wild-type 'FT' aligned only one nonsynonymous SNP to Bra010198; this gene is a homolog of Arabidopsis KNS4/UPEX1, which encodes a putative β-(1,3)-galactosyltransferase that controls pollen exine development. Comparative sequencing verified the SNP position on the fifth exon of Bra010198 in mutant ftms. Further genotyping revealed that the male sterile phenotype was fully co-segregated with this SNP. Quantitative real-time PCR indicated that Bra0101918 specifically expressed in stamen. The data presented herein suggested that Bra010198 is a strong candidate gene for ftms. Hence, we developed a male sterile line for potential application in breeding and expanded the knowledge about the molecular mechanism underlying male sterility in Chinese cabbage.

Pectin methylesterase inhibitor (PMEI) family can be related to male sterility in Chinese cabbage (Brassica rapa ssp. pekinensis).

Pectin-related genes play significant roles in pollen development and pollen tube growth, and their allelic variations are one of the major reasons for the abnormal development of male gametophyte. Currently, little is known about the role of the PMEI family in male sterility of plants. In this study, 97 putative PMEI genes were identified in Brassica rapa genome. By a phylogenetic analysis, the PMEI family was divided into 10 clades with highly conserved structural characteristics. The publically available RNA-seq data on different tissues of B. rapa accession Chiifu-401-42 revealed that 23 PMEI isoforms were flower-specific genes. We created a recessive genic male sterile mutant (ftms) in Chinese cabbage. This mutant was a doubled haploid line with stable inheritance, derived from Chinese cabbage 'FT' generated through a combination of radiation mutagenesis and isolated microspore culture. The transcriptome profiles of the floral buds of ftms and its wild-type line 'FT' were determined using RNA-seq. A total of 17 PMEI genes were found to be differentially expressed; all of them were down-regulated in ftms compared to their levels in 'FT'. Consistent with the transcriptome data, all these genes were observed to be highly expressed in the floral buds of 'FT' using qRT-PCR analysis. Of these, eight genes were specifically expressed in the floral buds of 'FT'; three of these (Bra019903, Bra014099, and Bra032239) were stamen-specific genes. The results contribute to further elucidation of the regulatory mechanisms underlying male sterility in Chinese cabbage.

Rapid alkalinization factor (RALF) genes are related to genic male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Male sterility is commonly and widely used for hybrid seed production in Brassica rapa L. ssp. pekinensis (B. rapa), and some genes related to anther and pollen development may play significant roles in pollen fertility. Hence, in this study, we performed whole-genome scanning and identified 38 genes from the RALF gene family in B. rapa, which were grouped into four clades according to the phylogenetic tree. Thirty-six of the sequences contained only exons; the other two contained both exonic and intronic sequences. Conserved motifs of the 38 RALF genes were identified. The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. Moreover, a total of 14 RALF-like differentially expressed genes (DEGs) were identified by RNA-Seq between male fertile and male sterile buds, and all were highly or specifically expressed in male fertile buds of the AB line ‘AB01’. A qRT-PCR analysis confirmed that the expression patterns of the 14 RALF DEGs in the AB line ‘AB01’ were similar to those in the other three genic male sterility (GMS) AB lines ‘AB02’, ‘AB03’, and ‘AB04’, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the results of qRT-PCR validated the RNA-Seq transcriptome as accurate and reliable. This study will provide valuable information for exploring the molecular mechanism underlying sterility in GMS AB lines of B. rapa.

Comparative transcript profiling of fertile and sterile flower buds from multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

We studied the underlying causes of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis) by identifying differentially expressed genes (DEGs) related to pollen sterility between fertile and sterile flower buds. In this work, we verified the stages of sterility microscopically and then performed transcriptome analysis of mRNA isolated from fertile and sterile buds using Illumina HiSeq2000 platform sequencing. Approximately 80% of ~229 million high-quality paired-end reads were uniquely mapped to the reference genome. In sterile buds, 699 genes were significantly up-regulated and 4096 genes were down-regulated. Among the DEGs, 28 pollen cell wall-related genes, 54 transcription factor genes, 45 phytohormone-related genes, 20 anther and pollen-related genes, 212 specifically expressed transcripts, and 417 DEGs located on linkage group A07 were identified. Six transcription factor genes BrAMS, BrMS1, BrbHLH089, BrbHLH091, BrAtMYB103, and BrANAC025 were identified as putative sterility-related genes. The weak auxin signal that is regulated by BrABP1 may be one of the key factors causing pollen sterility observed here. Moreover, several significantly enriched GO terms such as "cell wall organization or biogenesis" (GO:0071554), "intrinsic to membrane" (GO:0031224), "integral to membrane" (GO:0016021), "hydrolase activity, acting on ester bonds" (GO:0016788), and one significantly enriched pathway "starch and sucrose metabolism" (ath00500) were identified in this work. qRT-PCR, PCR, and in situ hybridization experiments validated our RNA-seq transcriptome analysis as accurate and reliable. This study will lay the foundation for elucidating the molecular mechanism(s) that underly sterility and provide valuable information for studying multiple-allele-inherited male sterility in the Chinese cabbage line 'AB01'.

Characterization and expression profiling of MYB transcription factors against stresses and during male organ development in Chinese cabbage (Brassica rapa ssp. pekinensis)

MYB proteins comprise a large family of plant transcription factors that play regulatory roles in different biological processes such as plant development, metabolism, and defense responses. To gain insight into this gene superfamily and to elucidate its roles in stress resistance, we performed a comprehensive genome-wide identification, characterization, and expression analysis of MYB genes in Chinese cabbage (Brassica rapa ssp. pekinensis). We identified 475 Chinese cabbage MYB genes, among which most were from R2R3-MYB (256 genes) and MYB-related (202) subfamilies. Analysis of sequence characteristics, phylogenetic classification, and protein motif structures confirmed the existence of several categories (1R, 2R, 3R, 4R, and 5R) of Chinese cabbage MYB genes, which is comparable with MYB genes of other crops. An extensive in silico functional analysis, based on established functional properties of MYB genes from different crop species, revealed 11 and four functional clades within the Chinese cabbage R2R3-MYB and MYB-related subfamilies, respectively. In this study, we reported a MYB-like group within the MYB-related subfamily contains 77 MYB genes. Expression analysis using low temperature-treated whole-genome microarray data revealed variable transcript abundance of 1R/2R/3R/4R/5R-MYB genes in 11 clusters between two inbred lines of Chinese cabbage, Chiifu and Kenshin, which differ in cold tolerance. In further validation tests, we used qRT-PCR to examine the cold-responsive expression patterns of 27 BrMYB genes; surprisingly, the MYB-related genes were induced more highly than the R2R3-MYB genes. In addition, we identified 10 genes with corresponsive expression patterns from a set of salt-, drought-, ABA-, JA-, and SA-induced R2R3-MYB genes. We identified 11 R2R3-MYBs functioning in resistance against biotic stress, including 10 against Fusarium oxysporum f.sp. conglutinans and one against Pectobacterium carotovoram subsp. caratovorum. Furthermore, based on organ-specific expression data, we identified nine R2R3-MYBs that were constitutively expressed in male reproductive tissue, which may provide an important key for studying male sterility in Chinese cabbage. The extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of MYB genes in stress resistance in addition to their growth regulatory functions, ultimately providing the basis for functional characterization and exploitation of the candidate MYB genes for genetic engineering of Chinese cabbage.

Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)

Mitochondrial-targeted Expression of <i>orf456</i> Causes Male Sterility in Chinese Cabbage (<i>Brassica rapa</i> L.)

Construction and analysis of a suppression subtractive hybridization (SSH) library of genic multiple-allele inherited male-sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Utilization of male sterility is a key method for producing crossbred Chinese cabbage (Brassica rapa L. ssp. pekinensis (Lour.) Olsson. In this study, suppression subtractive hybridization (SSH) was used to construct sterility and fertility cDNA libraries, which included differentially, expressed clones between fertile and sterile buds of the A/B line ‘AB01’. The positive clones were randomly selected by polymerase chain reaction amplification (PCR) and 25 high quality sequences (22 from the fertile-tester library and three from the sterile-tester libraries) were generated. The fragment lengths varied from 77 to 469 bp. Differential expression patterns between fertile and sterile buds were selected and verified using five expressed sequence tags (ESTs). Results indicated that, three ESTs were expressed only in fertile buds and two ESTs were down-regulated in sterile buds. According to the Basic Local Alignment Search Tool (BLAST) screening and functional annotation, the 25 ESTs were homologous to known sequences deposited in National Center for Biotechnology Information (NCBI). These genes had homology to known proteins such as flowers/buds development proteins, metabolic-related proteins, cell structure proteins, cell growth/division proteins and secondary metabolic-related proteins. The results suggested that, these proteins played a critical role in nuclear male sterility progression of genic multiple-allele inherited male-sterility in Chinese cabbage.

Isolation of fertility-related genes of multiple-allele-inherited male sterility in Brassica rapa ssp pekinensis by cDNA-AFLP

To better understand the molecular mechanisms of multiple-allele-inherited male sterility in Chinese cabbage (Brassica rapa ssp pekinensis), differentially expressed genes in fertile and sterile plants must be isolated. We used cDNA-AFLP analysis to isolate differentially expressed genes in fertile and sterile buds of the two-type line, AB01. Sixteen high-quality sequences were generated, 11 of which were up-regulated in fertile buds, and five of which were up-regulated in sterile buds. Based on BLAST screening and functional annotation, these genes have homology with genes encoding known flower- or bud-specific proteins, metabolism-related proteins and cell-structure proteins. In addition, the full-length cDNA sequences of the actin gene were cloned from the cabbage plants by RACE and used as an internal standard for semi-quantitative reverse transcription-PCR. Expression of three flower- or bud-specific differentially expressed transcript-derived fragments in fertile and sterile buds was examined using RT-PCR; the expression patterns of these genes were similar to the patterns observed in the cDNA-AFLP analysis.

Molecular cloning and characterization of BcMYBogu, a novel member of the MYB family involved in OguCMS in Brassica campestris ssp. chinensis

In the attempt to elucidate the molecular mechanism of CMS. Ogura cytoplasmic male sterile (OguCMS) lines were obtained in Chinese cabbage after interspecific hybridization between Brassica. napus L. OguCMS and B. campestris ssp. chinensis followed by recurrent backcross with B. campestris ssp. chinensis as the pollen donor. The CMS lines were significantly characterized by the whitish anther and indehiscence of anther. The tapetal hypertrophy with excess vacuola-tion was the first observed defective soon after the tetrad stage, subsequently the microspores defected in pollen wall forma-tion, and later the cytoplasm detached from the exine wall and underwent degeneration. With aid of cDNA-AFLP and RACE approaches, we cloned the BcMYBogu(GenBank accession No: EF127861) in Chinese cabbage, which is premature expressed in early and middle stage floral buds of OguCMS lines, and predicted to encode a novel protein with a DNA binding domain: SH[AL]QKY[RF] motif at the N-terminus. Phylogenetic comparison revealed that the BcMYBogu was clustered with AtMYB32, AtMYB26 and AtMYB4, which were indicated to be involved in male sterility in Arabidopsis thaliana. The BcMYBogu transcript was detected in rosette leaves, floral buds and stems by RT-PCR analysis. Compared with the maintainer, the expression level of BcMYBogu was increased in these organs, especially in floral buds of OguCMS lines. Our investigation suggests that BcMYBogu is a new member of the MYB family involved in male sterility in Chinese cabbage.

RNA Fingerprinting of the Differential Expression Fragments Related to Cytoplasmic Male Sterility in Chinese Cabbage

In order to investigate the differential expression of the genes related to cytoplasmic male sterility in Chinese cabbage (Brassica campestris L. ssp. Penkinsis), a modified RNA fingerprinting technique was developed to compare the difference in the total RNA from flower bud of Chinese cabbage among cytoplasmic male sterility (CMS) lines, maintainer lines and F1 hybrids. Four stably differential fragments S47-412, S93-622, S176-343 and S199-904 were amplified, cloned and sequenced with primers selected from 186 random primers. Based on the nucleotide sequence of the four differential fragments, four pairs of specific primers were designed to validate the differential fragments. The validation showed S47-412 and S93-622 were false positives and S176-343 and S199-904 were confirmed by PCR with the specific primers. Sequence analysis revealed that both of two differential fragments had strong homology with the nucleotide sequence of orf224/atp6 site of Polima CMS and the nucleotide sequence of S176-343 and S199-904 had a superposed region. All these indicate that the two fragments probably have strong relationship with cytoplasmic male sterility in Chinese cabbage.

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage.

Molecular markers for genic male sterility in Chinese cabbage

The gms gene of Chinese cabbage (Brassica campestris ssp. chinensis) conferring a recessivegenic male sterility was mapped with AFLPmarkers. Four markers were found to betightly linked to the gene with a distanceof <1 cM using a mapping population of327 individuals. The AFLPs were cloned andsequenced. Sequence derived primers yieldedclearly distinguishable PCR fragmentsrepresenting sequences tightly linked tothe gms gene. The sequence taggedsite (STS) markers can be used for markerassisted selection of male sterile plantsamong segregating populations.