Development of STS markers closely linked to the Ppd-H1 photoperiod response gene of barley (Hordeum vulgare L.)

A BC₂ population of 353 plants segregating for the Ppd-H1 photoperiod response gene was developed from a cross between the winter barley ’Igri’ and the spring barley ’Triumph.’ Bulk segregant analysis identified six AFLP markers closely linked to the Ppd–H1 gene and three strongly amplified AFLP bands that mapped 0.8-cM distal, 0.6-cM proximal and 2.3-cm proximal to Ppd-H1 were cloned and sequenced. Southern-blot analysis showed that the cloned fragments were single-copy sequences in ’Igri’, the variety from which they were derived. Two of the sequences were absent from ’Triumph’ while the third detected a single-copy sequence. The cloned fragments were used to design specific sequence tagged site (STS) primer pairs to assist in the construction of a high-resolution map of the Ppd-H1 region. Received: 22 March 2000 / Accepted: 10 April 2000     

An RFLP map of diploid Hordeum bulbosum L. and comparison with maps of barley (H. vulgare L.) and wheat (Triticum aestivum L.)

This paper describes the first extensive genetic map of Hordeum bulbosum, the closest wild relative of cultivated barley. H. bulbosum is valuable for haploid production in barley breeding, and because of desirable agronomic characteristics, it also has potential for trait introgression into barley. A H. bulbosum map will assist introgression and provide a basis for the identification of QTLs for crossability with barley and other potentially useful genes. The present study used a population of 111 individuals from a PB1×PB11 cross to develop a genetic linkage map of diploid H. bulbosum (2n=2x=14) based on barley, wheat and other ”anchor” cereal RFLP markers previously mapped in other species. Because of the cross-pollinating and highly polymorphic nature of H. bulbosum, up to four alleles showed segregation at any one locus, and five different segregation types were found. This enabled maps to be developed for the PB1 and PB11 parents, as well as a combined map. In total, 136 RFLP loci were mapped with a marker coverage of 621 cM. The markers were generally colinear with barley but H. bulbosum had less recombination in the centromeric regions and similar or more in the distal regions. Cytological studies on pollen mother cells at metaphase-I showed marked distal localization of chiasmata and a frequency consistent with the genetic map length. This study showed that H. bulbosum was highly polymorphic, making it suitable for trait analysis and supplementing maps of barley. Received: 20 November 2000 / Accepted: 5 January 2001     

RFLP mapping of BaYMV resistance gene rym3 in barley (Hordeum vulgare)

The rym3 (formerly designated ym3) gene conferring resistance to barley yellow mosaic virus (BaYMV) is effective against all strains of the virus but up to now has not been mapped to any chromosome. We performed a linkage analysis, using DNA extracted from individually harvested mature leaves of 153 F₂ plants derived from a cross between BaYMV-resistant cv ’Ishuku Shirazu’ carrying rym3 and susceptible cv ’Ko A’. Additionally, the F₃ lines derived from F₂ plants were grown in the BaYMV-infested field and examined for their reaction to BaYMV. Our results indicated that rym3 is located on the short arm of chromosome 5H and flanked by RFLP markers MWG28and ABG705A at distances of 7.2 and 11.7 cM, respectively. The chromosomal configuration estimated by DNA markers around rym3 and the utilization of these molecular markers for pyramiding with the BaYMV resistance genes in barley breeding programs are discussed. Received: 24 August 1998 / Accepted: 30 January 1999<@head-com-p1a.lf>Communicated by F. Salamini     

Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae

 Histological analysis was performed aimed at elucidating the origin and the developmental process of somatic embryos of two Brazilian cultivars of barley (Hordeum vulgare vulgare), 'MN-599' and 'A-05'. The observed site of somatic embryo origin (SSEO) could originate from a superficial callus cell, possibly indicating a unicellular origin, or from epidermal and subepidermal callus cells, representing a multicellular origin. A fold, the somatic embryo scutellum that subsequently develops into a cotyledonary leaf, indicates the somatic embryo differentiation. The somatic embryos also showed a growth increase of the primary root and, occasionally, a delay in root development. A possible alternative pathway for the origin of somatic embryos is suggested, in which a SSEO forms a clump of somatic embryos. Received: 4 June 1998 / Revision received: 28 August 1998 / Accepted: 7 December 1998     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

Identification of random amplified polymorphic DNA (RAPD) markers linked to the v locus in barley, Hordeum vulgare L.

 Recombinant backcross lines of barley were produced from a cross between Kanto Nakate Gold (KNG; two-rowed) and Azumamugi (AZ; six-rowed) after backcrosses of F₁ plants with AZ as the recurrent parent. Each of these lines had an introgressed segment from chromosome 2 of KNG. Two recombinant backcross lines, L1 and M3-13, were used for an initial screening of polymorphism. After screening a total of 888 oligonucleotides as arbitrary primers, we identified eight random amplified polymorphic DNAs (RAPDs) between backcross lines and AZ. Among the RAPD fragments, CMNA-38₇₀₀ was linked to the v locus with a recombination frequency of zero, while OPJ-09₈₅₀ and OPP-02₇₀₀ were linked to the v locus at a map distance of 1.4 cM. Thus, the three RAPD markers were clustered around the v locus since the lengths of introgressed chromosomal segments in the L1 and M3-13 lines were no less than 38 cM. The other five RAPD fragments that we identified were not linked to the v locus. Received: 14 January 1997 / Accepted: 14 February 1997     

QTL analysis of tolerance to a German strain of BYDV-PAV in barley (Hordeum vulgare L.)

One hundred and forty six barley doubled-haploid lines (DH lines) were tested for variation in grain yield, yield components, plant height, and heading date after artificial infection with a German isolate of barley yellow dwarf virus (BYDV-PAV-Braunschweig). Of these 146 lines 76 were derived from the cross of the barley yellow dwarf virus (BYDV) tolerant cultivar ’Post’ to cv ’Vixen’ (Ryd2) and 70 from the cross of Post to cv ’Nixe’. Phenotypic measurements were gathered on both non-infected plants and plants artificially inoculated with BYDV-PAV by viruliferous aphids in pot and field experiments for three years at two locations. For all traits a continuous variation was observed suggesting a quantitative mode of inheritance for tolerance against BYDV-PAV. Using skeleton maps constructed using SSRs, AFLPs and RAPDs, two QTLs for relative grain yield per plant after BYDV infection, explaining about 47% of the phenotypic variance, were identified in Post × Vixen at the telomeric region of chromosome 2HL and at a region containing the Ryd2 gene on chromosome 3HL. In Post × Nixe, a QTL was found in exactly the same chromosome 2HL marker interval. In this cross, additional QTL were mapped on chromosomes 7H and 4H and together these explained about 40% of the phenotypic variance. QTL for effects of BYDV infection on yield components, plant height, and heading date generally mapped to the same marker intervals, or in the vicinity of the QTL for relative grain yield, on chromosomes 2HL and 3HL, suggesting that these regions are of special importance for tolerance to the Braunschweig isolate of BYDV-PAV. Possible applications of marker-assisted selection for BYDV tolerance based on these results are discussed. Received: 1 December 2000 / Accepted: 9 March 2001     

Localising QTLs for leaf rust resistance and agronomic traits in barley (Hordeum vulgare L.)

The Hordeum vulgare accession ’HOR 1063’ was crossed with the barley cultivar Krona, and 220 doubled haploid lines were produced based on this cross. A molecular map was constructed based on RFLP markers. Field trials were performed over 2 years and at two locations. In field trials, resistance to leaf rust by means of artificial infection, heading date, plant height and Kernel weight were assessed. For leaf rust resistance, 4 QTLs were localised, that explained 96.1% of the genetic variation. One QTL on chromosome 4H confirmed a position found in another genetic background and one mapped to the same position as Rph16 on chromosome 2H. All digenic effects decreased the effects of the respective QTLs. In addition to the denso-locus and the hex-v locus, other QTLs influencing heading date, plant length and kernel weight were found in this cross. Received: 16 July 1999 / Accepted: 7 September 1999     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

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     

Genetic relationships among Stylosanthes species as revealed by sequence-tagged site markers

 Nineteen sequence-tagged site (STS) primer pairs were designed on coding and non-coding regions in nine published Stylosanthes genes, which were mostly derived from cDNA. Direct sequencing of PCR products derived from genomic DNA allowed us to identify introns and to design specific primers flanking these introns. The use of 24 STS primer pairs for the detection of intra- and inter-specific variation in Stylosanthes based on size differences was tested on a core set of Stylosanthes species. Based on these results, 20 STS markers were selected to determine genetic relationships among 63 genotypes representing 24 Stylosanthes species. A total of 148 alleles were amplified and analyzed, resulting in a genetic similarity value ranging from 0.62 to 0.98 among the species. Based on cluster analysis, three main groups and three subgroups were determined, and most of the species were classified unambiguously. Alloploid species were recognized by the occurrence of more than one allele per STS marker, indicating fixed heterozygosity. Sixteen STS markers were useful for the identification of genotypes within a species. Inter-species relationships, as revealed by STS, were in general agreement with previous morphological and molecular relationship studies. These STS markers are useful as an additional tool for the identification of species, subspecies and genotypes in Stylosanthes, with a view to plant conservation and breeding. Received: 2 June 1998 / Accepted: 28 October 1998     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

Strong extracellular nuclease activity displayed by barley (Hordeum vulgare L.) uninucleate microspores

 Electroporation is becoming an increasingly important technique for plant transformation. Nevertheless, no positive results were achieved in barley when uninucleate microspores were used as target cells. Since it was previously demonstrated that electric shocks create pores in the microspore cell wall, experiments were designed to verify the presence of nucleases in the electroporation mix. Aliquots of all the solutions used for microspore extraction, purification and transformation were collected and analysed using supercoiled pBI 221 as a substrate; a nuclease activity was detected in all samples. Though microspore rinsing removed most nucleolytic activity in the supernatants, DNA preservation in the electroporation buffer was difficult to achieve, because microspores appeared capable of synthesising and releasing endonucleases at any time. Microspore chilling at 0°C was fairly effective in reducing nuclease secretion in the mix, whereas 1%PEG or 10 mM EDTA maintained most of the DNA in a supercoiled or circular relaxed form. EDTA effects were counterbalanced by Mg²⁺, but not Ca²⁺ or Zn²⁺, and enhanced by Mn²⁺. Barley microspore nucleases actively degraded different DNAs as well as TMV RNA, and apparently had a molecular weight above 30 kDa. Nuclease inactivation with EDTA did not alter microspore viability and allowed a transient expression of the uidA gene in electroporated barley microspores. Received: 13 January 1997 / Accepted: 28 February 1997     

Use of locus-specific AFLP markers to construct a high-density molecular map in barley

 By using 25 primer combinations, 563 AFLP markers segregating in a recombinant inbred population (103 lines, F₉) derived from L94/Vada were generated. The 38 AFLP markers in common to the existing AFLP/RFLP combined Proctor/Nudinka map, one STS marker, and four phenotypic markers with known map positions, were used to assign present AFLP linkage groups to barley chromosomes. The constructed high-density molecular map contains 561 AFLP markers, three morphological markers, one disease resistance gene and one STS marker, and covers a 1062-cM genetic distance, corresponding to an average of one marker per 1.9 cM. However, extremely uneven distributions of AFLP markers and strong clustering of markers around the centromere were identified in the present AFLP map. Around the centromeric region, 289 markers cover a genetic distance of 155 cM, corresponding to one marker per 0.5 cM; on the distal parts, 906 cM were covered by 277 markers, corresponding to one marker per 3.3 cM. Three gaps larger than 20 cM still exist on chromosomes 1, 3 and 5. A skeletal map with a uniform distribution of markers can be extracted from the high-density map, and can be applied to detect and map loci underlying quantitative traits. However, the application of this map is restricted to barley species since hardly any marker in common to a closely related Triticum species could be identified. Received: 16 June 1997 / Accepted: 9 October 1997     

Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.)

 Boron toxicity has been recognised as an important problem limiting production in the low-rainfall regions of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in barley has been characterised but the mode of inheritance and the location of genes controlling tolerance were not previously known. A population of 150 doubled-haploid lines from a cross between a boron toxicity tolerant Algerian landrace, Sahara 3771, and the intolerant Australian cultivar Clipper was screened in four tolerance assays. An RFLP linkage map of the Clipper×Sahara population was used to identify chromosomal regions associated with boron tolerance in barley. Interval regression-mapping allowed the detection of four chromosomal regions involved in the boron tolerance traits measured. A region on chromosome 2H was associated with leaf-symptom expression, a region on chromosome 3H was associated with a reduction of the affect of boron toxicity on root growth suppression, a region on chromosome 6H was associated with reduced boron uptake, and a region on chromosome 4H was also associated with the control of boron uptake as well as being associated with root-length response, dry matter production and symptom expression. The benefits and potential of marker-assisted selection for boron toxicity tolerance are discussed. Received: 18 December 1997 / Accepted: 28 November 1998     

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents. Received: 5 March 1997 / Revision received: 4 September 1997 / Accepted: 13 September 1997     

Optimization of gene transfer into barley (Hordeum vulgare L.) mature embryos by tissue electroporation

 This study was conducted to detect the optimum conditions for DNA transfer into mature embryos of barley via electroporation. Cultured mature embryos of barley were directly electroporated in the presence of the pBI 121 vector carrying both the β-glucuronidase (GUS) and neomycin phosphotransferase II (npt II) genes. It was found that 500 v/cm and 500 μFd capacitance was the optimum combination for healthy germination of the transformed plants from mature electroporated embryos. Effects of culture duration before electroporation and selection antibiotic concentrations on germination were also examined. Gene transfer performed on 3-day-old cultures resulted in the highest germination frequencies. GUS expression was observed on transversal sections of embryos and mature leaves from 3 month-old regenerants. PCR and Southern blot analyses show the presence of the npt II transgene in the genome of a plant. Received: 15 June 1999 / Revision received: 27 September 1999 / Accepted: 26 October 1999     

Several QTLs involved in osmotic-adjustment trait variation in barley (Hordeum vulgare L.)

 Osmotic adjustment (OA) was previously demonstrated to be an important adaptive mechanism of drought tolerance in cereals. In order to determine which genomic regions are involved in OA variation, 187 barley (Hordeum vulgare L.) recombinant inbred lines (RILs) derived from a cross between Tadmor (drought tolerant) and Er/Apm (susceptible) were studied in a growth chamber for their OA capacity (through correlated traits and by calculation), at an early growth stage and under two water treatments (soil moisture of 14% and 100% of field capacity). The continuous distribution of the traits and their broad-sense line heritabilities, ranging from 0.04 to 0.44, indicated that OA and related traits should have a polygenic nature. A subset of 167 RILs were also genotyped using 78 RFLP, 32 RAPD and three morphological markers and a linkage map was constructed. Despite strong environmental effects acting on the traits, interval mapping and single-marker ANOVA allowed the detection of three QTLs for relative water content (RWC), four QTLs for osmotic potential (ψ_π), two QTLs of osmotic potential at full turgor (ψ_π100) and one QTL for osmotic adjustment at a soil moisture of 14% field capacity. For the irrigated treatment, only two QTLs were detected: one for RWC and one for ψ_π100. Two chromosomal regions were involved in several OA-related trait variations and could be considered as regions controlling OA; these were present on chromosome 1 (7H) and chromosome 6 (6H), whereas other regions were specific for one trait. No major QTL was found. However, the genomic region involved in OA-related traits on chromosome 1 (7H) in barley seemed to be conserved for OA variation among cereals. Epistatic effects, with or without additive effects, acted on the traits. Received: 15 July 1997 / Accepted: 29 October 1997     

HvPIP1;6, a barley (Hordeum vulgare L.) plasma membrane water channel particularly expressed in growing compared with non-growing leaf tissues

The aim of the present study was to identify water channel(s) which are expressed specifically in the growth zone of grass leaves and may facilitate growth-associated water uptake into cells. Previously, a gene had been described (HvEmip) which encodes a membrane intrinsic protein (MIP) and which is particularly expressed in the base 1 cm of barley primary leaves. The functionality of the encoding protein was not known. In the present study on leaf 3 of barley (Hordeum vulgare L.), a clone was isolated, termed HvPIP1;6, which has 99% amino acid sequence identity to HvEmip and belongs to the family of plasma membrane intrinsic proteins (PIPs). Expression of HvPIP1;6 was highest in the elongation zone, where it accounted for >85% of expression of known barley PIP1s. Within the elongation zone, faster grower regions showed higher expression than slower growing regions. Expression of HvPIP1;6 was confined to the epidermis, with some expression in neighboring mesophyll cells. Expression of HvPIP1;6 in Xenopus laevis oocytes increased osmotic water permeability 4- to 6-fold. Water channel activity was inhibited by pre-incubation of oocytes with 50 microM HgCl(2) and increased following incubation with the phosphatase inhibitor okadaic acid or the plant hormone ABA. Plasma membrane preparations were analyzed by Western blots using an antibody that recognized PIP1s. Levels of PIP1s were highest in the elongation and adjacent non-elongation zone. The developmental expression profile of HvPIP2;1, the only known barley water channel belonging to the PIP2 subgroup, was opposite to that of HvPIP1;6.     

Towards second-generation STS (sequence-tagged sites) linkage maps in conifers: a genetic map of Norway spruce ( Picea abies K.)

Genetic linkage maps have been produced for a wide range of organisms during the last decade, thanks to the increasing availability of molecular markers. The use of microsatellites (or Simple Sequence Repeats, SSRs) as genetic markers has led to the construction of “second-generation” genetic maps for humans, mouse and other organisms of major importance. We constructed a second-generation single-tree genetic linkage map of Norway spruce (Picea abies K.) using a panel of 72 haploid megagametophytes with a total of 447 segregating bands [366 Amplified Fragment Length Polymorphisms (AFLPs), 20 Selective Amplification of Microsatellite Polymorphic Loci (SAMPLs) and 61 SSRs, each single band being treated initially as a dominant marker]. Four hundred and thirteen markers were mapped in 29 linkage groups (including triplets and doublets) covering a genetic length of 2198.3 cM, which represents 77.4% of the estimated genome length of Picea abies (approximately 2839 cM). The map is still far from coalescing into the expected 12 chromosomal linkage groups of Norway spruce (2n = 2x = 24). A possible explanation for this comes from the observed non-random distribution of markers in the framework map. Thirty-eight SSR marker loci could be mapped onto 19 linkage groups. This set of highly informative Sequence Tagged Sites (STSs) can be used in many aspects of genetic analysis of forest trees, such as marker-assisted selection, QTL mapping, positional cloning, gene flow analysis, mating system analysis and genetic diversity studies. Received: 5 November 1997 / Accepted: 16 March 1998