Assignment of DNA markers to Nicotiana sylvestris chromosomes using monosomic alien addition lines

 A sesquidiploid hybrid (PPS, 2n=32) between Nicotiana plumbaginifolia (PP, 2n=20) and N. sylvestris (SS, 2n=24) was backcrossed to N. plumbaginifolia to produce monosomic alien addition lines. A total of 89 2n=21 plants, each containing two sets of N. plumbaginifolia chromosomes and a single N. sylvestris chromosome, were obtained in the BC₁ and BC₂ generations. These plants were classified into 12 groups based on morphological characteristics. The N. sylvestris chromosomes in these plants were identified by RFLP and karyotype analyses. Among the 84 probes tested, 20 could not detect N. sylvestris-specific DNA bands, and the remaining 64 were assigned to 9 normal and 6 aberrant synteny groups. The 9 normal synteny groups corresponded to chromosomes 2, 4, 5, 6, 7, 8, 9, 10 and 12, respectively. Four aberrant synteny groups were the result of chromosome translocations, and 2 were deletions. Received: 10 April 1996 / Accepted: 5 July 1996     

Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines

Seven monosomic addition plants, each containing the full complement of Nicotiana plumbaginifolia (2n = 20, genome constitution PP) and an aberrant chromosome of Nicotiana sylvestris (2n = 24, SS), were produced from backcrosses of hyperdiploid derivatives of the sesquidiploid hybrid PPS to N. plumbaginifolia. The N. sylvestris chromosomes in these plants were characterized by karyotype analysis, Southern hybridization with DNA markers previously localized on N. sylvestris chromosomes and a 269-bp fragment from the 3' end of 25S rDNA, and fluorescence in situ hybridization using 25S rDNA, 5S rDNA and telomere repeats (TTTAGGG)_n as probes. The N. sylvestris chromosomes in these plants were identified to be telocentrics 6S, 7S and 8S, and deletions 7S, 10, 12S and 12L, respectively. The successful identification of aberrant chromosomes in these lines enabled us to assign DNA markers to arms and sub-arm regions of N. sylvestris chromosomes. All aberrant chromosomes in the addition lines could be transmitted through mitosis and meiosis. The potential applications of the addition lines in high-resolution physical mapping, the isolation of N. sylvestris chromosomes by flow cytometry, and an understanding of the chromosomal distribution of 45S rDNA in N. sylvestris are discussed.     

Construction of a high-density SNP genetic map in flue-cured tobacco based on SLAF-seq

Tobacco (Nicotiana tabacum L., 2n = 48) is an important agronomic crop and model plant. Flue-cured tobacco is the most important type and accounts for approximately 80 % of tobacco production worldwide. The low genetic diversity of flue-cured tobacco impedes the construction of a high-density genetic linkage map using simple sequence repeat (SSR) markers and warrants the exploitation of single nucleotide polymorphic (SNP) markers from genomic regions. In this article, initially using specific locus-amplified fragment sequencing, we discovered 10,891 SNPs that were subsequently used as molecular markers for genetic map construction. Combined with SSR markers, a final high-density genetic map was generated containing 4215 SNPs and 194 SSRs distributed on 24 linkage groups (LGs). The genetic map was 2662.43 cM in length, with an average distance of 0.60 cM between adjacent markers. Furthermore, by mapping the SNP markers to the ancestral genomes of Nicotiana tomentosiformis and Nicotiana sylvestris, a large number of genome rearrangements were identified as occurring after the polyploidization event. Finally, using this novel integrated map and mapping population, two major quantitative trait loci (QTLs) were identified for flue-curing and mapped to the LG6 of tobacco. This is the first report of SNP markers and a SNP-based linkage map being developed in tobacco. The high-density genetic map and QTLs related to tobacco curing will support gene/QTL fine mapping, genome sequence assembly and molecular breeding in tobacco.     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture. Received: 17 November 1999; in revised form: 3 February 2000 / Accepted: 3 February 2000     

Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n=2x=16) yellow-flowered Medicago sativa ssp. quasifalcata and the diploid (2n=2x=16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning > 659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n=1x=1.0 x 10⁹ bp) gives a ratio of < 1500 kb per centimorgan.     

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.     

A Genetic Map of Lettuce (Lactuca sativa L.) with Restriction Fragment Length Polymorphism, Isozyme, Disease Resistance and Morphological Markers

A detailed linkage map of lettuce was constructed using 53 genetic markers including 41 restriction fragment length polymorphism (RFLP) loci, five downy mildew resistance genes, four isozyme loci and three morphological markers. The genetic markers were distributed into nine linkage groups and cover 404 cM which may be 25-30% of the lettuce genome. The majority (31 of 34) of the RFLP probes detected single segregating loci, although seven of these may have been homologous to further monomorphic loci. When several loci were detected by a single probe, the loci were generally linked, suggesting tandem duplications. One probe, however, detected loci in three linkage groups suggesting translocations. The five downy mildew resistance genes (Dm1, Dm3, Dm4, Dm5/8 and Dm13), segregating in the Calmar x Kordaat cross, represented each of the four resistance gene linkage groups. Dm5/8 is flanked by two cDNA loci, each located 10 cM away. These flanking markers will be used to study the source of variation in downy mildew genes and are also part our strategy to clone resistance genes.     

Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A₂ and A₃ subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

A comparative map of wild rice (Zizania palustris L. 2n=2x=30)

We present the first genetic map of wild rice (Zizania palustris L., 2n=2x=30), a native aquatic grain of northern North America. This map is composed principally of previously mapped RFLP (restriction fragment length polymorphism) genetic markers from rice (Oryza sativa 2n=2x=24). The map is important as a foundation for genetic and crop improvement studies as well as a reference for genome organization comparisons among species of Gramineae. A comparative mapping approach with rice is especially useful because wild rice is grouped in the same subfamily, Oryzoideae, and no other mapping comparison has yet been made within the subfamily. As rice is the reference point for mapping and gene cloning in cereals, establishing a consensus map within the subfamily identifies conserved and unique regions. The genomes of wild rice and rice differ in total DNA content (wild rice has twice that of rice) and the number of chromosome pairs (wild rice=15 versus rice=12). The wild rice linkage map reported herein consists of 121 RFLP markers on 16 linkage groups spanning 1805 cM. Two linkage groups consist of only two markers. Colinear markers were found representing all rice linkage groups except #12. The majority of rice loci mapped to colinearly arranged arrays in wild rice (92 of 118). Features of the map include duplication of portions of three rice linkage groups and three possible translocations. The map gives basic information on the composition of the wild rice genome and provides tools to assist in the domestication of this important food source. Received: 25 August 1998 / Accepted: 20 February 1999     

An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants

DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Comparative RFLP mapping of meadow and tall fescue

 Molecular markers based on restriction fragment length polymorphism (RFLP) were used to construct a genetic linkage map in diploid meadow fescue, Festuca pratensis Huds. (2n=2x=14, genomic designation PP), and to compare its genomic relationship with a related species, hexaploid tall fescue (Festuca arundinacea Schreb.; 2n=6x=42, PPG₁G₁G₂G₂). Using a collection of 66 tall-fescue (heterologous) markers, an RFLP linkage map was constructed in F. pratensis. This map, which has a total length of 280.1 cM, includes seven linkage groups. A comparison of 33 markers that were mapped in both F. pratensis and F. arundinacea detected highly conserved linkage groups between these two species. Our data are consistent with the proposal that one of the genomes of F. arundinacea was derived from F. pratensis. However, since significant changes in marker sequences, map distances, and homoeologous linkage groups were also detected between the two species, it appears that the P genome diverged substantially during evolution from the diploid to the hexaploid Festuca. Received: 23 May 1997 / Accepted: 15 January 1998     

A comparative map of wild rice (Zizania palustris L. 2n=2x=30)

We present the first genetic map of wild rice (Zizania palustris L., 2n=2x=30), a native aquatic grain of northern North America. The map is composed principally of previously mapped RFLP (restriction fragment length polymorphism) genetic markers from rice (Oryza sativa 2n=2x=24). The map is important as a foundation for genetic and crop improvement studies, as well as a reference for genome organization comparisons among Gramineae species. A comparative mapping approach with rice is especially useful because wild rice is grouped in the same subfamily, Oryzoideae, and no other mapping comparison has yet been made within the subfamily. As rice is the reference point for mapping and gene cloning in cereals, establishing a consensus map within the subfamily identifies conserved and unique regions. The genomes of wild rice and rice differ in total DNA content (wild rice has twice that of rice) and chromosome pairs (wild rice=15 versus rice=12). The wild rice linkage map reported herein consists of 121 RFLP markers on 16 linkage groups spanning 1805 cM. Two linkage groups consist of only two markers. Colinear markers were found representing all rice linkage groups except #12. The majority of rice loci mapped to colinearly arranged arrays in wild rice (92 of 118). Features of the map include duplication of portions of three rice linkage groups and three possible translocations. The map gives basic information on the composition of the wild rice genome and provides tools to assist in the domestication of this important food source. Received: 25 August 1998 / Accepted: 20 February 1999 (Corrected version. Originally published in TAG 99:793–799)     

RFLP genetic linkage maps from four F(2.3) populations and a joinmap of Gossypium hirsutum L

An RFLP genetic linkage joinmap was constructed from four different mapping populations of cotton (Gossypium hirsutum L.). Genetic maps from two of the four populations have been previously reported. The third genetic map was constructed from 199 bulk-sampled plots of an F_2.3 (HQ95–6×’MD51ne’) population. The map comprises 83 loci mapped to 24 linkage groups with an average distance between markers of 10.0 centiMorgan (cM), covering 830.1 cM or approximately 18% of the genome. The fourth genetic map was developed from 155 bulk-sampled plots of an F_2.3 (119– 5 sub-okra×’MD51ne’) population. This map comprises 56 loci mapped to 16 linkage groups with an average distance between markers of 9.3 cM, covering 520.4 cM or approximately 11% of the cotton genome. A core of 104 cDNA probes was shared between populations, yielding 111 RFLP loci. The constructed genetic linkage joinmap from the above four populations comprises 284 loci mapped to 47 linkage groups with the average distance between markers of 5.3 cM, covering 1,502.6 cM or approximately 31% of the total recombinational length of the cotton genome. The linkage groups contained from 2 to 54 loci each and ranged in distance from 1.0 to 142.6 cM. The joinmap provided further knowledge of competitive chromosome arrangement, parental relationships, gene order, and increased the potential to map genes for the improvement of the cotton crop. This is the first genetic linkage joinmap assembled in G. hirsutum with a core of RFLP markers assayed on different genetic backgrounds of cotton populations (Acala, Delta, and Texas plain). Research is ongoing for the identification of quantitative trait loci for agronomic, physiological and fiber quality traits on these maps, and the identification of RFLP loci lineage for G. hirsutum from its diploid progenitors (the A and D genomes). Received: 23 February 2001 / Accepted: 8 June 2001     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

Chloroplast segregation in somatic hybrids of Nicotiana plumbaginifolia and N. sylvestris having different ratios of parental nuclear genomes

Summary Fusion of mesophyll protoplasts of haploid Nicotiana plumbaginifolia (P) and N. sylvestris (S) resulted in the production of somatic hybrid plants of various ploidy levels. Analysis of the restriction fragment patterns of chloroplast DNA from 118 plants belonging to genome constitutions PS, PPS, PSS, and PPSS revealed that two had a pattern corresponding to a mixture of parental DNA while all the others had the pattern of either N. plumbaginifolia or N. sylvestris. In the latter case, the ratio of the two parental types fits 1∶1 in all the four genome constitutions studied. Since the protoplasts used in the fusion experiment were physiologically similar and the hybrid cells were not deliberately selected, these results suggest that chloroplast segregation in the somatic hybrids is independent of the chloroplast input of the fusion partners and the nuclear background of the fusion products.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.