Relationship between parental chromosomic contribution and nuclear DNA content in the coffee interspecific hybrid C. pseudozanguebariae×C. liberica var ‘dewevrei’

 F₁ hybrids were obtained between two coffee species with the same chromosome number (2n=22) but with different nuclear DNA contents [C. pseudozanguebariae (PSE) 2C=1.13 pg and C. liberica var ‘dewevrei’ (DEW) 2C=1.42 pg]. G2 hybrids were obtained by open-pollination of the F₁ hybrids. Genomic in situ hybridisation (GISH) and flow cytometry were used on six F₁ hybrids and seven G2 hybrids to determine their parental chromosomic contribution and their nuclear DNA content (qDNA), respectively. GISH efficiently identified chromosomes from both species. F₁ hybrids had a qDNA intermediate between that of the parental species and contained the expected 11 chromosomes from each species. There was a linear relationship between the number of PSE chromosomes and the nuclear DNA content, which indicates that flow cytometry can be used to give a rough estimate of the parental chromosomic contribution in G2 hybrids. Received: 1 August 1997/Accepted: 25 August 1997     

Non-random inheritance of organellar genomes in symmetric and asymmetric somatic hybrids between Lycopersicon esculentum and L. pennellii

Summary The organization of the mitochondrial genome and the genotype of the chloroplast genome was characterized using restriction fragment length polymorphisms in a population (82 individuals) of symmetric and asymmetric somatic hybrids of tomato. The protoplast fusion products were regenerated following the fusion of leaf mesophyll protoplasts of Lycopersicon esculentum (tomato cv UC82) with suspension cell protoplasts of L. pennellii that had been irradiated with 5, 10, 15, 25, 50, or 100 kRads from a gamma source. The chloroplast genome in the somatic hybrids showed a random pattern of inheritance, i.e., either parental genome was present in equal numbers of regenerants, while in asymmetric somatic hybrids, the chloroplast genotype reflected the predominant nuclear genotype, i.e., tomato. The mitochondrial genome in the symmetric somatic hybrids showed a non-random pattern of inheritance, i.e., predominantly from the L. pennellii parent; asymmetric somatic hybrids had more tomato-specific mitochondrial sequences than symmetric somatic hybrids. The non-random inheritance of the chloroplast and mitochondrial DNA in these tomato protoplast fusion products appears to be influenced by the nuclear background of the regenerant.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC₃) and selfed (S₃) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC₃ plants (originated from somatic hybrids 3, 4, 10) with 2–8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.     

A genome-wide analysis of wide compatibility in rice and the precise location of the S5 locus in the molecular map

 The discovery of wide-compatibility varieties (WCVs) that are able to produce normal fertility hybrids when crossed both to indica and japonica rice has enabled the fertility barrier between indica and japonica subspecies to be broken and provided the possibility of developing inter-subspecific hybrids in rice breeding programs. However, a considerable variation in the fertility level of hybrids from the same WCV crossed to different varieties has often been observed. One hypothesis for this variable fertility is that additional genes are involved in hybrid fertility besides the wide-compatibility gene (WCG). To assess such a possibility, we performed a genome-wide analysis by assaying a large population from a three-way cross ‘02428’/‘Nanjing 11’//‘Balilla’ using a total of 171 RFLP probes detecting 191 polymorphic loci distributed throughout the entire rice linkage map. Our analysis recovered 3 loci conferring significant effects on hybrid fertility. The major locus on chromosome 6 coincided in chromosomal location with the previously identified S ₅ locus, and the 2 minor loci that mapped to chromosomes 2 and 12, respectively, were apparently distinct from all previously reported hybrid sterility genes. Interaction between the indica and japonica alleles at each of the loci caused a reduction in hybrid fertility. The joint effect of the 2 minor loci could lead to partial sterility even in the presence of the WCG. The location of the S ₅ locus on the molecular marker linkage map was determined to be approximately 1.0 cM from the RFLP locus R2349. This tight linkage will be useful for marker-aided transfer of the WCG in hybrid rice breeding and for map-based cloning. Received: 5 February 1997 / Accepted: 4 April 1997     

Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers

 Linkage maps for the apple cultivars ‘Prima’ and ‘Fiesta’ were constructed using RFLP, RAPD, isozyme, AFLP, SCAR and microsatellite markers in a ‘Prima’בFiesta’ progeny of 152 individuals. Seventeen linkage groups, putatively corresponding to the seventeen haploid apple chromosomes, were obtained for each parent. These maps were aligned using 67 multi-allelic markers that were heterozygous in both parents. A large number of duplicate RFLP loci was observed and, in several instances, linked RFLP markers in one linkage group showed corresponding linkage in another linkage group. Distorted segregation was observed mainly in two regions of the genome, especially in the male parent alleles. Map positions were provided for resistance genes to scab and rosy leaf curling aphid (Vf and Sd ₁, respectively) for the fruit acidity gene Ma and for the self-incompatibility locus S. The high marker density and large number of mapped codominant RFLPs and some microsatellite markers make this map an ideal reference map for use in other progenies also and a valuable tool for the mapping of quantitative trait loci. Received: 17 November 1997 / Accepted: 9 December 1997     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

GISH,AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange [Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2n=4x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.The first two authors contributed equally to this paper.     

Hybrid origin of some ornamentals of Allium subgenus Melanocrommyum verified with GISH and RAPD

 Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) methods have been used to verify the hybridogenic origin and to identify the parental species of some ornamental cultivars in the subgenus Melanocrommyum of the genus Allium. The cultivars had been selected from seed obtained after uncontrolled pollination in breeders’ fields. The combination of GISH analysis with RAPD markers is very suitable for testing the hybridogenic origin of plants and to ascertain the parental species of the hybrids in such cases. As suspected, A. macleanii and A. cristophii are the parental species of ‘Globemaster’. The parental species of cultivar ‘Globus’ are A. karataviense and A. stipitatum, and not A. cristophii and A. giganteum as has been assumed on morphological grounds. Cultivars ‘Lucy Ball’ and ‘Gladiator’ are of hybrid origin, though only one of the parental species, A. hollandicum, could be confirmed. The cultivars ‘Purple Sensation’, ‘Mount Everest’, ‘White Giant’, ‘Michael H. Hoog’ and ‘Mars’ are not hybrids since neither GISH nor RAPD suggest the presence of a second genome. ‘Purple Sensation’ belongs to A. hollandicum, ‘Mount Everest’, ‘White Giant’ and ‘Mars’ to A. stipitatum,‘Michael H. Hoog’ to A. rosenorum. Received: 3 July 1997 / Accepted: 9 October 1997     

Development of a wheat genotype combining the recessive crossability alleles kr1kr1kr2kr2 and the 1BL.1RS translocation, for the rapid enrichment of 1RS with new allelic variation

The main objective of the present work was to develop a wheat genotype containing both the recessive crossability alleles (kr1kr1kr2kr2), allowing high crossability between 6x wheat and diploid rye, and the 1BL.1RS wheat/rye translocation chromosome. This wheat genotype could be used as a recipient partner in wheat–rye crosses for the efficient introduction of new allelic variation into 1RS in translocation wheats. After crossing the wheat cultivars ‘Mv Magdaléna’ and ‘Mv Béres’, which carry the 1BL.1RS translocation involving the 1RS chromosome arm from ‘Petkus’, with the line ‘Mv9 kr1’, 117 F₂ plants were analysed for crossability, ten of which had higher than 50% seed set with rye and thus presumably carried the kr1kr1kr2kr2 alleles. Four of the ten plants contained the 1BL.1RS translocation in the disomic condition as detected by genomic in situ hybridization (GISH). The wheat × rye F₁ hybrids produced between these lines and the rye cultivar ‘Kriszta’ were analysed in meiosis using GISH. 1BL.1RS/1R chromosome pairing was detected in 62.4% of the pollen mother cells. The use of fluorescent in situ hybridization (FISH) with the repetitive DNA probes pSc119.2, Afa family and pTa71 allowed the 1R and 1BL.1RS chromosomes to be identified. The presence of the 1RS arm from ‘Kriszta’ besides that of ‘Petkus’ was demonstrated in the F₁ hybrids using the rye SSR markers RMS13 and SCM9. In four of the 22 BC₁ progenies analysed, only ‘Kriszta’-specific bands were observed with these markers, though the presence of the 1BL.1RS translocation was detected using GISH. It can be concluded that recombination occurred between the ‘Petkus’ and ‘Kriszta’ 1RS chromosome arms in the translocated chromosome in these plants.     

Origin of an alien disomic addition with an aberrant homologue of chromosome-10 of tomato and its meiotic behaviour in a potato background revealed through GISH

 While characterising potato (Solanum tuberosum, 2n=4x=48) clones with alien tomato (Lycopersicon esculentum) chromosome additions, a single addition for chromosome-10 of tomato was identified through restriction fragment length polymorphism (RFLP) analysis. This plant, 2101–1, was a BC₂ derivative from a cross between a potato (+) tomato fusion hybrid backcrossed to potato. Cytological analysis of its somatic chromosomes through genomic in situ hybridisation (GISH) indicated the presence of four genomes of potato with two alien tomato chromosomes, of which one was much smaller than the other. Analysis of chromosome pairing at the pachytene and metaphase-I stages of microsporogenesis indicated that the large and small chromosomes were homologues. Thus, it was a disomic addition for chromosome-10 of tomato. The size difference was found to be due to a deletion. Fluorescent in situ hybridisation (FISH) experiments, using the telomeric repeat pAtT4 from Arabidopsis thaliana and the sub-telomeric repeat TGRI, showed intact telomeres and sub-telomeres for both alien chromosomes. Thus, the deletion that the smaller of the homologues suffered was interstitial and most probably occurred in the centromeric heterochromatic region of the long arm. The pattern of distribution of large and small chromosomes to telophase-II nuclei during microsporogenesis indicated that the deletion did not affect the meiotic behaviour of the smaller chromosome. In contrast, the frequencies of transmission of the large and the small chromosomes through the female parent, estimated in 96 BC₃ progeny of plants by RFLP and GISH analyses, appeared to be very different, 69.2% and 3.8% respectively. This study also provides evidence that two different chromatids of a pair of homologues, rather than two chromatids of a single chromosome, are most likely to be involved in the origin of a disomic. The aberrant chromosome can be used for the physical mapping of chromosome-10. Received: 9 June 1998 / Accepted: 28 October 1998     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Direct evidence for high level of autosyndetic pairing in hybrids of Thinopyrum intermedium and Th. ponticum with Triticum aestivum

 Genomic in situ hybridization (GISH) was used to distinguish autosyndetic from allosyndetic pairing in the hybrids of Thinopyrum intermedium and Th. ponticum with Triticum aestivum cv ‘Chinese Spring’ (CS). All hybrids showed high autosyndetic pairing frequencies among wheat chromosomes and among Thinopyrum chromosomes. The high autosyndetic pairing frequencies among wheat chromosomes in both hybrids suggested that Th. intermedium and Th. ponticum carry promoters for homoeologous chromosome pairing. The higher frequencies of autosyndetic pairing among Thinopyrum chromosomes than among wheat chromosomes in both hybrids indicated that the relationships among the three genomes of Th. intermedium and among the five genomes of Th. ponticum are closer than those among the three genomes of T. aestivum. Received: 19 September 1996 / Accepted: 18 April 1997     

Transmission of alien tomato chromosomes from BC1 to BC2 progenies derived from backcrossing potato (+) tomato fusion hybrids to potato: the selection of single additions for seven different tomato chromosomes

 By backcrossing three BC₁ genotypes of potato (+) tomato fusion hybrids to different tetraploid potato pollinators, BC₂ populations were produced. A combined total of 97 BC₂ plants from three BC₂ populations were analysed with chromosome-specific probes through restriction fragment length polymorphism (RFLP) for the presence of alien tomato chromosomes. The number of different alien tomato chromosomes transmitted through the female BC₁ parent ranged from 0 to 6, and the average number of different alien chromosomes transmitted per BC₂ plant varied between 1.7 and 3.4 in the different populations. This variation corresponded to the chromosome constitution of the individual BC₁ parents: parent 6739, which possessed 11 different alien chromosomes in a single condition, gave rise to progeny with a lower average number of alien chromosomes per plant than the BC₁ parent 2003 that possessed 2 of the 12 alien chromosomes in a disomic condition. In the latter case, the higher transmission rate was attributed to the more regular distribution of the two alien chromosomes in the disomic condition because of regular bivalent formation during meiosis as revealed by genomic in situ hybridisation (GISH) and fluorescent in situ hybridisation (FISH). The transmission frequencies of individual alien chromosomes were subjected to statistical analysis to test whether the maternal genotypes had an effect on alien-chromosome transmission. Among the BC₂ plants, a total of 27 single additions were detected for as many as seven different chromosomes (1, 2, 4, 6, 8, 10 and 12) out of the 12 possible types. Received: 4 March 1997 / Accepted: 28 August 1997     

The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance

Five intergeneric hybrids between the chrysanthemum cultivar ‘Zhongshanjingui’ (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While ‘Zhongshanjingui’ bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids’ flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from ‘Zhongshanjingui’ and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.     

Analysis of hybrids of durum wheat with Thinopyrum junceiforme using RAPD markers

 The objective of this study was to detect the presence of alien chromatin in intergeneric hybrids of durum wheat (Triticum turgidum, 2n=4x=28; AABB genomes) with the perennial grass Thinopyrum junceiforme (2n=4x=28; J₁J₁J₂J₂) using RAPD markers. The first step was to identify amplification of species-specific DNA markers in the parental grass species and durum wheat cultivars. Initially, the genomic DNA of five grass species (Thinopyrum junceiforme, Th. bessarabicum, Lophopyrum elongatum, Leymus karataviensis and Elytrigia pycnantha) and selected durum cultivars (‘Langdon’, ‘Durox’, ‘Lloyd’, ‘Monroe’, and ‘Medora’) was screened with 40 oligonucleotide primers (nano-mers). Three oligonucleotides that amplified DNA fragments specific to a grass species or to a durum cultivar were identified. Primer PR21 amplified DNA fragments specific to each of the five durum cultivars, and primers PR22 and PR23 amplified fragments specific to each of the grass species. Intergeneric hybrids between the durum cultivars ‘Langdon’, ‘Lloyd’ and ‘Durox’ and Th. junceiforme, and their backcross (BC) progeny were screened with all 40 primers. Six primers amplified parent-specific DNA fragments in the F₁ hybrids and their BC₁ progeny. Three primers, PR22, PR23 and PR41, that amplified Th. junceiforme DNA fragments in both F₁ and BC₁ were further analyzed. The presence of an amplified 1.7-kb Th. junceiforme DNA fragment in the F₁ hybrids and BC₁ progeny was confirmed using Southern analysis by hybridization with both Th. junceiforme genomic DNA and Th. junceiforme DNA amplified with primer PR41. With the exception of line BC₁F₂ no. 5, five selfed progeny of BC₁ and a BC₂ of line 3 (BC₁F₂ no. 3בLloyd’) from a cross of ‘Lloyd’×Th. junceiforme showed the presence of the 1.7-kb DNA fragment. All selfed BC₁ and BC₂ lines retained the 600-bp fragment that was confirmed after hybridization with Th. junceiforme DNA amplified with primer PR22. Other experiments using RFLP markers also showed the presence of up to seven Th. junceiforme DNA fragments in the F₁ hybrids and their BC progeny after hybridization with Th. junceiforme DNA amplified with primer PR41. These studies show the usefulness of molecular markers in detecting alien chromatin/DNA fragments in intergeneric hybrids with durum wheat. Received: 21 November 1996 / Accepted: 21 March 1997     

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     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998