Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Molecular and cytogenetic characterization of an Oryza officinalis–O. sativa chromosome 4 addition line and its progenies

The wild species Oryza officinalis Wall. ex Watt (2n = 24, CC) is a valuable genetic resource for rice (O. sativa L., 2n = 24, AA) breeding and genomics research. Genomic in situ hybridization (GISH) and molecular approaches were used to determine the nature and composition of the additional chromosome in a monosomic alien addition line (MAAL) of O. officinalis and its backcross progenies. The extra wild species chromosome in the MAAL (2n = 2x = 25) was a mosaic one, comprising of the long arm of chromosome 4 from O. officinalis and the short arm from O. sativa. Comparative analysis showed that O. sativa and O. officinalis shared high synteny of restriction fragment length polymorphism (RFLP) markers and low synteny of simple sequence repeat (SSR) markers. A DNA methylation alteration was revealed at C619 in the MAAL and progenies. Analysis of progenies of the MAAL indicated that introgression segments were small in size and introgression was not evenly distributed along the long arm. One recombination hot spot between C513 and RG177 was identified, which is in a gene-rich region.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Characterization of an Alien Chromosome of <Emphasis Type="Italic">Oryza officinalis</Emphasis> Transferred the Genomic and Cytological Environment of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Distant hybridization between rice species and their genome interactions have become the hot points of genetic research. The new formed hybrid was unstable and many of the alien chromosomes would be excluded out in the meiosis procedure. In this study, we investigated the phenomenon that the monosomic alien addition lines (MAALs) of Oryza officinalis (CC)–Oryza sativa (AA) inherited in the genomic and cytoplasmic environments of cultivated rice. In a series of backcross programs, every time the MAALs were backcrossed with the recurrent parent Hejiang19, we get a portion of the backcross progenies that were similar to the female parent MAALs in morphological characters (with rolled leaves) and chromosome constitutions (2n + 1). The monosomic alien chromosome exhibited stable inheritance in the genomic and cytoplasmic environments of cultivated rice. Molecular biology and fluorescence in situ hybridization (FISH) analyses on mitotic cells showed that the alien chromosomes in MAALs were inclined to be transferred integrated into the backcross progenies with rolled leaves and no big fragments of alien chromosomes introgressing into the chromosomes of cultivated rice. By FISH analysis on the meiotic cells, we proved that the single alien chromosome performing nonstandard meiosis and behaving like a dispensable genetic factor, which would be the cytological reasons for the stable inheritance of alien chromosomes.     

Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice,O. sativa L.

Summary Sterile AC hybrids between cultivated Oryza sativa (AA) and a distant wild species, O. officinalis (CC), were backcross to O. sativa. Most of the BC₁ progenies were allotriploid (AAC), a few were hypotriploid. AAC progenies were again backcrossed to O. sativa. BC₂ progenies consisting of disomic or aneuploid individuals were examined for the presence of O. officinalis traits. Eleven different traits from O. officinalis were identified in these progenies. Segregation data in the subsequent generations suggest that these traits are monogenic in nature. Two of these genes — for resistance to BPH and WBPH — are of value in rice improvement. The extremely low recovery of recombinant progenies is in agreement with the very low amount of pairing between A and C genomes. Because of this restricted recombination, the genotype of the recurrent parent was reconstituted after two backcrosses only. Thus, the BC₂ progenies look remarkably similar to O. sativa. Most of them are stable and fertile and also interfertile with other O. sativa breeding lines. Some of the BPH-and WBPH-resistant progenies are comparable in yield to the best O. sativa parents and are being evaluated as varietal possibilities.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Isolation of a new retrotransposon-like DNA sequence and its use in analysis of diversity within the Oryza officinalis

To better understand the genetic diversity of the wild relatives of rice (Oryza sativa L.) in the O. officinalis species complex repetitive DNA markers were obtained from the diploid species of this complex. One cloned sequence from O. eichingeri gave intense hybridization signals with all species of the O. officinalis complex. This 242 bp clone, named pOe.49, has a copy number from 0.9 to 4.0 × 10⁴ in diploid species of this complex. Analysis of the primary structure and database searches revealed homology of pOe.49 to a number of sequences representing part of the integrase coding domain of retroviruses and gypsy-like retrotransposons. Sequencing of specific PCR products confirmed that pOe.49 is part of a gypsy-like retrotransposon. RFLP analysis was used to study the genomic organisation of pOe.49 among 30 accessions of the O. officinalis complex using 10 restriction enzymes. Diversity analysis based on 120 polymorphic fragments obtained from the RFLP assay grouped the O. officinalis complex accessions by genome, species and eco-geographic groups. The results suggest that, with further characterization, this retrotransposon-like DNA sequence may be useful for phylogenetic analysis of species in the O. officinalis complex. This revised version was published online in August 2006 with corrections to the Cover Date.     

GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines

Two Brassica napus--Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F₂ progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Identification of genome constitution of Oryza malampuzhaensis, O. minuta,and O. punctata by multicolor genomic in situ hybridization

Multicolor genomic in situ hybridization (McGISH) was applied to identify the genomic constitution of three tetraploid species (2n = 4x = 48) in the Oryza officinalis complex of the genus Oryza, i.e. Oryza malam-puzhaensis, Oryza minuta, and Oryza punctata. The genomic probes used were from three diploids, i.e. Oryza officinalis (CC), Oryza eichingeri (CC) and Oryza punctata (BB), respectively. The results indicated that all three tetraploids are allotetraploid with the genomic constitution of BBCC, and among them the genome constitution of O. malampuzhaensis was verified for the first time. Restoration of the independent taxonomic status of O. malampuzhaensis is suggested. One pair of satellite chromosomes belonging to the B genome was identified in O. malampuzhaensis, but no such satellite chromosomes were found in either O. minuta or the tetraploid O. punctata. The average chromosome length of the C genome was found to be slightly larger than that of the B-genome chromosomes of O. minuta, but not in the tetraploids O. punctata and O. malampuzhaensis. McGISH also revealed that the B genome of O. minuta and the B genome of diploid O. punctata showed clear differentiation from each other. Therefore, the suggestion was proposed that the B genome in diploid O. punctata was not the source of the B genome of O. minuta. The present results proved that multicolor GISH had high resolution in identifying the genomic constitution of polyploid Oryza species. Received: 14 February 2000 / Accepted: 13 November 2000     

Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv. to rice,Oryza sativa L.

Oryza latifolia, a tetraploid wild relative of cultivated rice is an important source of resistance to bacterial blight (BB), the brown planthopper (BPH) and the whitebacked planthopper (WBPH). Interspecific hybrids were obtained between an elite breeding line (IR31917-45-3-2) of Oryza sativa (2n=24 AA) and O. latifolia Acc. No. 100914 (2n=48 CCDD). The crossability in F1 was 7.58% and it ranged from 0.11 to 0.62 in backcross generations. The F1 hybrid showed 2-6 II, 0-2 III, 0-1 IV and 22-32 I; the mean being 3.92 II + 0.11 III + 0.02 IV + 27.30 I per cell at diakinesis. Monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. latifolia were characterized based on morphology and isozyme banding pattern. The MAALs were designated as MAAL-1, MAAL-2, MAAL-4, MAAL-5, MAAL-6, MAAL-7, MAAL-8, MAAL-9, MAAL-10, MAAL-11 and MAAL-12. The female transmission rates of the alien chromosome varied from 4.4 to 35.5%, whereas 8 of the 11 MAALs transmitted the alien chromosome through the male gamete, the range being 1.7% (MAAL 10) to 11.9% (MAAL 12). Disomic progenies in BC3 and BC4 generations had complete resemblance to the O. sativa parent. Of the 2,295 disomic BC3F3 progenies, 309 showed introgression for resistance to BPH and 188 each for WBPH and BB resistance. Four plant progenies which were resistant to both BPH and WBPH were also resistant to BB race 2 of the Philippines. Nine of the 34 BC3F1 plants showed introgression for ten allozymes of O. latifolia, such as Est5, Amp1, Pgi1, Mdh3, Pgi2, Amp3, Pgd2, Est9, Amp2 and Sdh1, located on 8 of the 12 chromosomes. Alien introgression was also detected for morphological traits such as long awns, earliness, black hull, purple stigma and apiculus. Abnormal plants with many wild-species traits suddenly appeared in normal disomic progenies. These plants showing instability and abnormal segregation behaviour are being investigated for the activation of transposons.     

Characterization of euploid backcross progenies derived from interspecific hybrids between Oryza sativa and O. eichingeri by restriction fragment length polymorphism (RFLP) analysis and genomic in situ hybridization (GISH).

Restriction fragment length polymorphism (RFLP) analysis and GISH (genomic in situ hybridization) were performed on euploid plants derived from crosses between Oryza sativa (2n = 24, AA) and two brown planthopper-resistant accessions of O. eichingeri (2n = 24, CC). After screening with 164 RFLP markers, 60 of the 67 euploid plants were identified as introgression lines, each carrying 1-6 small O. eichingeri segments integrated on chromosomes 1, 2, 6, or 10. In the somatic chromosome preparations of F1 hybrid, O. eichingeri chromosomes, fluorescing greenish-yellow in the sequential GISH, appeared to be longer and to contain more heterochromatin than O. sativa ones, and this karyotypic polymorphism can be used to detect some introgressed O. eichingeri segments in euploid plants. In addition, GISH identification presented direct evidence for the transfer of small segments from O. eichingeri to O. sativa chromosome(s) which were subsequently recognized according to their condensation pattern, arm ratio, and chromosome length. The present results would contribute to the molecular mapping and selection of O. eichingeri--derived brown planthopper-resistant gene and positive yield QTLs.     

Comparative studies of isozymes in Oryza sativa,O. minuta,and their interspecific derivatives: evidence for homoeology and recombination

Enzyme electrophoresis was used to compare the isozyme phenotypes of Oryza sativa, IR31917 (AA genome), and two O. minuta accessions (Om 101089 and Om101141; BBCC genome) for ten enzyme systems. Between the two species, two systems were monomorphic (isocitrate dehydrogenase and alcohol dehydrogenase) and eight were polymorphic (shikimate dehydrogenase, phosphogluconate dehydrogenase, phosphoglucose isomerase, malate dehydrogenase, glutamate oxaloacetate transaminase, esterase, aminopeptidase, and endopeptidase). Polymorphism between O. minuta accessions was detected for shikimate dehydrogenase and glutamate oxaloacetate. As expected, the quaternary structure of the O. minuta isozymes was comparable to that of O. sativa. Possible allelic relationships with known O. sativa alleles and their genomic designation are discussed. Combined with chromosome data, the interspecific variation was exploited to monitor the relative genetic contribution of the two parents in the IR31917/Om101141 F₁ hybrids and recurrent (IR31917) backcross progenies. The isozyme content of F₁ hybrid reflected its triploid nature (ABC genome composition), while that of the backcross progenies paralleled the duplication of the A genome and the gradual loss of O. minuta chromosomes during the backcrossing process. Evidence is provided for a degree of homoeology between the A, B, and C genomes, and for introgression from O. minuta into O. sativa.     

Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.     

Infection with the parasitic angiosperm Striga hermonthica influences the response of the C3 cereal Oryza sativa to elevated CO2

Upland rice (Oryza sativa L.) was grown at both ambient (350 μmol mol^?1) and elevated (700 μmol mol^?1) CO₂ in either the presence or absence of the root hemi‐parasitic angiosperm Striga hermonthica (Del) Benth. Elevated CO₂ alleviated the impact of the parasite on host growth: biomass of infected rice grown at ambient CO₂ was 35% that of uninfected, control plants, while at elevated CO₂, biomass of infected plants was 73% that of controls. This amelioration occurred despite the fact that O. sativa grown at elevated CO₂ supported both greater numbers and a higher biomass of parasites per host than plants grown at ambient CO₂. The impact of infection on host leaf area, leaf mass, root mass and reproductive tissue mass was significantly lower in plants grown at elevated as compared with ambient CO₂. There were significant CO₂ and Striga effects on photosynthetic metabolism and instantaneous water‐use efficiency of O. sativa. The response of photosynthesis to internal [CO₂] (A/C_i curves) indicated that, at 45 days after sowing (DAS), prior to emergence of the parasites, uninfected plants grown at elevated CO₂ had significantly lower CO₂ saturated rates of photosynthesis, carboxylation efficiencies and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) contents than uninfected, ambient CO₂‐grown O. sativa. In contrast, infection with S. hermonthica prevented down‐regulation of photosynthesis in O. sativa grown at elevated CO₂, but had no impact on photosynthesis of hosts grown at ambient CO₂. At 76 DAS (after parasites had emerged), however, infected plants grown at both elevated and ambient CO₂ had lower carboxylation efficiencies and Rubisco contents than uninfected O. sativa grown at ambient CO₂. The reductions in carboxylation efficiency (and Rubisco content) were accompanied by similar reductions in nitrogen concentration of O. sativa leaves, both before and after parasite emergence. There were no significant CO₂ or infection effects on the concentrations of soluble sugars in leaves of O. sativa, but starch concentration was significantly lower in infected plants at both CO₂ concentrations. These results demonstrate that elevated CO₂ concentrations can alleviate the impact of infection with Striga on the growth of C₃ hosts such as rice and also that infection can delay the onset of photosynthetic down‐regulation in rice grown at elevated CO₂.     

<Emphasis Type="Italic">Cucumis</Emphasis> monosomic alien addition lines: morphological,cytological, and genotypic analyses

Cucumis hystrix Chakr. (HH, 2n=24), a wild relative of the cultivated cucumber, possesses several potentially valuable disease-resistance and abiotic stress-tolerance traits for cucumber (C. sativus L., CC, 2n=14) improvement. Numerous attempts have been made to transfer desirable traits since the successful interspecific hybridization between C. hystrix and C. sativus, one of which resulted in the production of an allotriploid (HCC, 2n=26: one genome of C. hystrix and two of C. sativus). When this genotype was treated with colchicine to induce polyploidy, two monosomic alien addition lines (MAALs) (plant nos. 87 and 517: 14 CC+1 H, 2n=15) were recovered among 252 viable plants. Each of these plants was morphologically distinct from allotriploids and cultivated cucumbers. Cytogenetic and molecular marker analyses were performed to confirm the genetic constitution and further characterize these two MAALs. Chromosome counts made from at least 30 meristematic cells from each plant confirmed 15 nuclear chromosomes. In pollen mother cells of plant nos. 87 and 517, seven bivalents and one univalent were observed at diakinesis and metaphase I; the frequency of trivalent formation was low (about 4–5%). At anaphase I and II, stochastic and asymmetric division led to the formation of two gamete classes: n=7 and n=8; however, pollen fertility was relatively high. Pollen stainability in plant no. 87 was 86.7% and in plant no. 517 was 93.2%. Random amplified polymorphic DNA analysis was performed using 100 random 10-base primers. Genotypes obtained with eight primers (A-9, A-11, AH-13, AI-19, AJ-18, AJ-20, E-19, and N-20) showed a band common to the two MAAL plants and C. hystrix that was absent in C. sativus, confirming that the alien chromosomes present in the MAALs were derived from C. hystrix. Morphological differences and differences in banding patterns were also observed between plant nos. 87 and 517 after amplification with primers AI-5, AJ-13, N-12, and N-20, suggesting that these plants may contain different C. hystrix chromosomes.Communicated by H. Nybom     

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