Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. maritima

 The present study shows that the recently described mitochondrial H haplotype is associated with cytoplasmic male-sterility (CMS). This new source of CMS appears to be different from the mitotype E-associated CMS most frequently found in natural populations. A mitotype H progeny with a sexual phenotype segregation was used to identify a gene restoring male fertility (R1H ). Using bulk segregant analysis (BSA), nine RAPD markers linked to this restorer locus were detected and mapped. The comparison with other Beta genetic maps shows that the closest RAPD marker, distant from R1H by 5.2 cM, belongs to the same linkage group as the monogermy locus. In order to determine the position of R1H more precisely, four RFLP loci within this linkage group were mapped in the segregating progeny. It thus became possible to construct a linkage map of the region containing the RFLP, RAPD and R1H loci. The closest RFLP marker was located 1.7 cM away from R1H. However, a nuclear gene restoring the ‘Owen’ CMS which is currently used in sugar beet breeding is reportedly linked to the monogermy locus, raising the question of a possible identity between the new CMS system and the ‘Owen’ CMS. Received: 15 September 1997 / Accepted: 1 December 1997     

Genetic basis of low-temperature-sensitive sterility in indica-japonica hybrids of rice as determined by RFLP analysis

 Low-temperature-sensitive sterility (LTSS) has become one of the major obstacles in indica-japonica hybrid rice breeding. In this study, we determined, using RFLP markers, the genetic basis of LTSS in two populations derived from crosses between indica and japonica parents, the BC₁F₁ of 3037/02428//3037 and the F₂ of 3037/02428. The fertility segregation in the two populations under low-temperature conditions was used as a measurement of the temperature sensitivity of the various genotypes in the populations. A RFLP survey of bulked extremes from the BC₁F₁ population identified three genomic regions, two on chromosome 1 and one on chromosome 12, that were likely to contain genes for LTSS (or Ste loci). One-way ANOVA and QTL analysis using a total of 19 markers from these three genomic regions resolved three Ste loci in the BC₁F₁ population and two Ste loci in the F₂ population. On the basis of chromosomal location these loci were distinct from those governing wide-compatibility identified in previous studies. Two- and three-way ANOVA showed that these loci acted essentially independent of each other in conditioning LTSS. The main mode of gene action was an interaction between the indica and the japonica alleles within each locus. For each respective locus this resulted in a drastic fertility reduction in the heterozygote state relative to the homozygote state. The results have significant implications in indica-japonica hybrid rice breeding programs. Received : 10 April 1996 / Accepted: 2 June 1997     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999     

Genetic characterization of weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes and RAPD markers

 Weedy rice (Oryza sativa L.) is an important resource for breeding and for studying the evolution of rice. The present study was carried out to identify the genetic basis of the weedy rices distributed in various countries of the world. One hundred and fifty two strains of weedy rice collected from Bangladesh, Brazil, Bhutan, China, India, Japan, Korea, Nepal, Thailand and the USA were tested for variations in six morpho-physiological characteristics and in 14 isozyme loci. Twenty six weedy strains selected from the above materials were assayed for the Est-10 locus, six RAPD loci of the nuclear genome, and one chloroplast locus. From the results of multivariate analysis based on the morpho-physiological characteristics and the isozymes, weedy rice strains were classified into indica and japonica types, and each type was further divided into forms resembling cultivated and wild rice. Thus, four groups designated as I, II, III and IV were identified. Weedy strains of group I (indica-type similar to cultivars) were distributed mostly in temperate countries, group II (indica-type similar to wild rice) in tropical countries, group III (japonica-type similar to cultivars) in Bhutan and Korea, group IV ( japonica-type similar to wild rice) in China and Korea. In group I, classified as indica, several strains showed japonica-specific RAPD markers, while some others had japonica cytoplasm with indica-specific RAPD markers in a heterozygous state at several loci. One weedy strain belonging to group II showed a wild rice-specific allele at the Est-10 locus. However, in groups III and IV, no variation was ound either for the markers on Est-10 or for the RAPD loci tested. Judging from this study, weedy rice of group I might have originated at least partly from gene flow between indica and japonica, whereas that of group II most probably originated from gene flow between wild and cultivated indica rice. Weedy rice of group III is thought to have originated from old rice cultivars which had reverted to a weedy form, and that of group IV from gene flow between japonica cultivars and wild rice having japonica backgrounds. Received: 2 May 1996 / Accepted: 30 August 1996     

RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat

Tan spot, caused by Pyrenophora tritici-repentis, is an economically important disease in major wheat production areas. The fungus can produce two genetically distinct symptoms on leaves of susceptible wheat genotypes: tan necrosis (nec) and extensive chlorosis (chl). Our objectives were to determine the number of genes conditioning resistance to tan spot in a population of wheat recombinant inbred lines, and map the chromosomal location of the resistance genes using RFLPs. Conidia produced by the P. tritici-repentis isolate Pti2 (nec+chl+) were used to inoculate seedlings of 135 recombinant inbred lines derived from the cross of the synthetic hexaploid wheat W-7984 with Opata 85. A subset of the population was inoculated with conidia produced by the isolates D308 (nec−chl+) and 86-124 (nec+chl−). Inoculated seedlings were rated on a scale of 1 to 5 based on lesion type. Necrosis-inducing culture filtrate produced by the isolate 86-124 was also used to screen the entire population. A map consisting of 532 markers was employed to identify significant associations between marker loci and tan spot resistance. The entire population was insensitive to culture filtrate produced by the isolate 86-124, and the entire subset was resistant to conidial inoculation of the same isolate. The population segregated for reaction to isolates D308 and Pti2, indicating that this population segregates for resistance to extensive chlorosis only, and not to tan necrosis. RFLP analysis indicated the presence of a gene with a major effect in 1AS, a gene with a minor effect in 4AL, and an interaction between the 1AS gene and a gene in 2DL. Together, these loci explained 49.0% of the variation in this population for resistance to tan spot produced by the isolate Pti2. Two regions one in 1BL and one in 3BL, were significantly associated with resistance to extensive chlorosis, but were not significant in the multiple regression model. It should be feasible to introgress these resistance loci into adapted genetic backgrounds by using a marker-assisted selection scheme. Received: 30 March 1996 / Accepted: 31 May 1996     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Detection of somaclonal variation in cultured rice cells using digoxigenin-based random amplified polymorphic DNA

A procedure for the non-radioactive detection of random amplified polymorphic DNA (RAPD) was developed and designated as digoxigenin (DIG)-based RAPD. Using this procedure, we analyzed somaclonal variation in cultured cells of rice. Somaclonal variation was found to increase with culture age. More than 50 polymorphic fragments were identified with the four primers tested. Random sequencing of 10 clones generated one intron, one 5′-noncoding, and eight non-redundant expressed sequences. A database search for homology showed that the eight exon sequences displayed a significant similarity to sequences already stored in EMBL, GenBank and DDBJ. The sources of the known genes ranged from microorganism to human, including three rice genes. The results showed that somaclonal variation might have occurred in transfer RNA, ribosomal protein, and other genes during cell culture. Received: 14 November 1997 / Revision received: 21 August 1998 / Accepted: 31 August 1998     

Identifying different types of de-differentiated microspores from indica-japonica F1 hybrids with subspecies-differentiating RFLP probes in rice

The indica, japonica and intermediary types of de-differentiated microspores from indica-japonica hybrids were identified with 11 subspecies-differentiating RELP probes in rice (Oryza sativa L.). The results showed that the distribution of indica, japonica and intermediary types of de-differentiated microspores could be easily detected in a simple and quick way using the RFLP method. Moreover, the microspores from the same hybrid but inoculated onto different media, or microspores from different hybrids when inoculated onto the same medium, often displayed distinctive distribution curves of de-differentiated microspores types, indicating that the media employed in this experiment had high selectivity for the de-differentiation of certain types of microspores. The application of the RELP method to de-differentiated microspore identification is of great theoretical and practical significance in rice doubled-haploid breeding. Received: 27 February 1996 / Accepted: 14 June 1996     

Molecular mapping of a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley (Hordeum vulgare L.)

The Rfm1a gene restores the fertility of msm1 cytoplasmic male-sterile lines in barley. We identified three RAPD markers linked to the Rfm1 locus (CMNB-07/800, OPI-18/900, and OPT-02/700) using isogenic lines and segregating BC₁F₁ and F₂ populations. Using a previously developed linkage map of barley, we located CMNB-07/800 and OPT-02/700 beside MWG2218 on chromosome 6HS. The linkage between MWG2218 and the Rfm1 locus was demonstrated using the segregating BC₁F₁ and F₂ populations. To confirm the chromosomal locations of these markers, we converted them to STSs and tested against two sets of wheat–barley chromosome addition lines. These STS markers, CMNB-07/800, OPT-02/700, and MWG2218, were amplified only in the addition lines possessing the chromosome 6H, thereby providing additional evidence the Rfm1 locus is located on chromosome 6H. Homoeologous relationships among fertility restoration genes in Triticeae are discussed. Received: 27 March 2000 / Accepted: 25 June 2000     

Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques

The discovery and application of the thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid seed production technology in rice. Use of the TGMS system in two-line breeding is simple, inexpensive, efficient, and eliminates the limitations associated with the cytoplasmic-genetic male sterility (CMS) system. An F₂ population developed from a cross between a TGMS indica mutant, TGMS–VN1, and a fertile indica line, CH1, was used to identify molecular markers linked to the TGMS gene and to subsequently determine its chromosomal location on the linkage map of rice. Bulk segregant analysis was performed using the AFLP technique. From the survey of 200 AFLP primer combinations, four AFLP markers (E2/M5–600, E3/M16–400, E5/M12–600, and E5/M12–200) linked to the TGMS gene were identified. All the markers were linked to the gene in the coupling phase. All except E2/M5–200 were found to be low-copy sequences. However, the marker E5/M12–600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64×Azucena and CT9993×IR62666, available at IRRI, Philippines, and Texas Tech University, respectively. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12–600, was sequenced so that a PCR marker can be developed for the marker-assisted transfer of this gene to different genetic backgrounds. The new TGMS gene is tentatively designated as tms4(t). Received: 13 July 1999 / Accepted: 27 July 1999     

RAPD and RFLP markers tightly linked to the locus controlling carnation (Dianthus caryophyllus) flower type

 Flower doubleness as a breeding characteristic is of major importance in carnation (Dianthus caryophyllus), one of the major cut-flowers sold worldwide, since flower architecture is of the utmost value in ornamentals. Based on the number of petals per flower, carnations are grouped into “single”, “semi-double” and “double” flower types. The first have five petals and are easily distinguishable, but of no economic value to the carnation industry. Flowers of standard and spray varieties, which constitute the largest market share, are usually of the double and semi-double type, respectively. These flower types are not easily distinguishable due to phenotypic overlaps caused by environmental conditions. To study the inheritance of this trait, several progeny segregating for flower type were prepared. Based on the number of single-flower type fullsibs among the offspring, we found that this phenotype is expressed only in plants homozygous for the recessive allele and that a dominant mutation in this allele causes an increase in petal number. Using random decamer primers, we identified a random amplified polymorphic DNA (RAPD) marker which is tightly linked to this recessive allele. The RAPD marker was cloned and used to generate a restriction fragment length polymorphic (RFLP) marker. This RFLP marker could discriminate with 100% accuracy between the semi-double and double- flower phenotypes in carnations of both Mediterranean and American groups. The advantages of RFLP over RAPD markers and their applicability to markerassisted selection in carnation are discussed. Received: 11 August 1997 / Accepted: 22 August 1997     

Reproducibility of the differential amplification between leaf and root DNAs in soybean revealed by RAPD markers

 Random amplified polymorphic DNA (RAPD) was used to determine whether such markers can be employed for detecting genomic modification during plant development or under certain stress environments. Pairwise comparisons in RAPD patterns of leaf and root DNA amplifications were studied for 11 soybean accessions representing different origins. Hydroponic culture was used for the ease of harvesting roots. From a total of 40 primers screened, it was found that 16 can detect leaf DNA polymorphism, 19 for root DNA polymorphism, while 10 show a greater consistency for detecting polymorphism between leaf and root (L/R) DNAs. Nevertheless, problems were encountered when the newly synthesized oligo-primers and different thermal cyclers were used to check the data. Several factors were then tested for their reproducibility. The results indicated that the amplified differences between root and leaf DNAs are mostly not affected by template DNA concentrations. The addition of DMSO (dimethyl sulphoxide) or TMAC (tetramethyl-ammonium chloride) also did not mask the L/R differences. However, DNA polymerase and oligo-primers synthesized from different manufacturers, as well as the thermal cyclers, reacted differently sometimes. Regardless of the general problems of reproducibility in RAPD patterns, some amplified differences remain between the L/R DNAs. The most distinct patterns involve differences in the relative intensity of amplified bands. Differential amplification might have occurred during plant leaf and root development. Southern hybridization of the eluted polymorphic bands against restriction digestion of total genomic DNA confirms their being homologous to soybean DNA fragments. Polymorphism of these specific L/R differences also exists among varieties. RAPD should be a useful tool in detecting genomic alterations during plant development or under certain stress environments, as long as the factors affecting the reproducibility of RAPD patterns can be properly controlled. An additional cycle of selection would be possible if such a type of polymorphism is proved to be correlated with certain developmental characters. Received: 7 October 1996 / Accepted: 20 May 1997     

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.)

 Bulked segregant analysis and comparative mapping were applied to identify molecular markers linked to the Rfo restorer gene used for the Ogu-INRA cytoplasmic male-sterility system in rapeseed. These markers were then used to localise the radish introgression on the B. napus genetic map constructed from the cross ‘Darmor.bzh’ x ’Yudal’. The introgression mapped on the DY15 linkage group. From the comparison of this latter group to the linkage group constructed on a F₂ progeny segregating for the radish introgression, it was concluded that the introgression had occurred through homoeologous recombination, that it was not distal and that it had replaced a B. napus region of around 50 cM. A QTL involved in aliphatic seed glucosinolate content was located on the DY15 linkage group at a position corresponding to one end of the introgression. The DNA markers identified in this study are being used in map-based cloning of the Rfo gene and in marker-assisted selection. Received: 3 December 1997 / Accepted: 17 December 1997     

Analyzing quantitative trait loci for yield using a vegetatively replicated F2 population from a cross between the parents of an elite rice hybrid

Although F₂s are the most informative populations for genetic analysis, it has been difficult to use F₂ populations directly for QTL analysis because it is usually difficult to assess the reliability of the data, due to an inability to estimate the experimental errors. In this study, we performed a QTL analysis for yield and yield-component traits of an F₂ population based on data from replicated field trials over 2 years using vegetative shoots of ratooned plants, making use of the ratooning habit of rice. The objective of this study was to explore the possibility of conducting QTL analyses directly based on an F₂ population by means of ratooning plants. The experimental population was from a cross between ’Zhenshan 97’ and ’Minghui 63’, the parents of ’Shanyou 63’, an elite rice hybrid widely grown in China. A genetic linkage map containing 151 molecular markers was constructed for QTL mapping. A total of 20 distinct QTLs were detected; eight of these were detected in both years and remaining 12 in only 1 year. Compared with the results of our previous analysis of the F_2:3 families from the same cross, it was shown that most of the QTLs detected in the ratooned F₂ population were also detected in the F_2:3 population. However, the estimates of both additive and dominant types of genetic effects for many of the QTLs based on F₂ ratoons were substantially larger than those based on F_2:3 families. The results indicate that vegetatively ratooned F₂ populations may have considerable utility in the mapping of QTLs, especially if dominant types of gene actions are of concern, although there were certain technical limitations in making use of such populations in the experiments. Received: 11 November 1999 / Accepted: 24 November 1999     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

RFLP and RAPD markers linked to the rosy leaf curling aphid resistance gene (Sd1) in apple

 Sd ₁ is a dominant gene for resistance to biotypes 1 and 2 of the rosy leaf curling aphid, Dysaphis devecta Wlk., which can cause economic damage to apple trees. This report describes the identification of three RFLP and four RAPD markers linked to Sd ₁ in a cross between the D. devecta susceptible variety ‘Prima’ (sd ₁ sd ₁) and the resistant variety ‘Fiesta’ (Sd ₁ sd ₁). Potted trees were artificially infested in the glasshouse, and the ratio of resistant:susceptible plants supported the hypothesis that the resistance was under the control of a single dominant gene. The position of the gene was mapped to a single locus on a ‘Fiesta’ chromosome, within 2 cM of three tightly linked RFLP markers (MC064a, 2B12a and MC029b); the four RAPD markers were located further away (between 13 and 46 cM). This is the first report of molecular markers for an aphid resistance gene in tree fruit crops. The potential application of these markers in a marker-assisted resistance breeding programme is discussed. Received: 1 July 1996/Accepted: 23 August 1996     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998