Loss of heterozygosity at individual loci in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> regenerants cultured with para-fluorophenylalanine

Precise chromosome segregation is vital for speciation and hybrid formation. The aim of this work was to study the chromosomes behavior and inheritance of maternal and paternal genomes in Arabidopsis regenerants obtained from in vitro cultured cells on the medium with para-fluorophenyalanine (PFPA). The Arabidopsis thaliana model hybrid between Columbia and Landsberg erecta ecotypes was developed, which chromosomes were easy to distinguish using the 12 SSLP selected markers. Also, the influence of PFPA on callus formation and regeneration of plants was analyzed. 20 regenerated plants cultured with PFPA were derived, three of which were shown to loss the heterozygosity in six loci by DNA markers analysis. Different models are certainly required to understand how and when the mechanisms leading to proper chromosome segregation are established in species and hybrids.     

Molecular mapping of the centromeres of tomato chromosomes 7 and 9

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F₂ populations of 1620Lycopersicon esculentum × L. pennellii (E × P) plants and 1640L. esculentum × L. pimpinellifolium (E × PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E × PM) and remotely related (E × P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in theL. esculentum × L. pennellii F₂ population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.     

Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Chromosome engineering: power tools for plant genetics

The term "chromosome engineering" describes technologies in which chromosomes are manipulated to change their mode of genetic inheritance. This review examines recent innovations in chromosome engineering that promise to greatly increase the efficiency of plant breeding. Haploid Arabidopsis thaliana have been produced by altering the kinetochore protein CENH3, yielding instant homozygous lines. Haploid production will facilitate reverse breeding, a method that downregulates recombination to ensure progeny contain intact parental chromosomes. Another chromosome engineering success is the conversion of meiosis into mitosis, which produces diploid gametes that are clones of the parent plant. This is a key step in apomixis (asexual reproduction through seeds) and could help to preserve hybrid vigor in the future. New homologous recombination methods in plants will potentiate many chromosome engineering applications.     

Influence of genetic background and heterozygosity on meiotic recombination in Arabidopsis thaliana.

Plant breeding relies on genetic variability generated by meiotic recombination. Control of recombination frequencies is not yet possible, but would significantly extend the options for plant-breeding strategies. A prerequisite would be variability of recombination frequencies. In this study, 15 transgenic kanamycin (KR) and hygromycin (HR) resistance gene insertions mapping to the five Arabidopsis thaliana chromosomes were used as genetic markers. Recombination frequencies were determined from the frequencies of resistance phenotypes within populations segregating for linked KR and HR markers. Recombination frequencies of marker pairs were compared among these four ecotypes, among F1s in both reciprocal forms derived from these ecotypes, and between F1s and their parent lines. On average, the recombination frequencies in F1 crosses were substantially higher (up to 2-fold) than in the homozygous parental ecotypes. A strong negative correlation between genetic similarities of ecotypes and recombination frequencies was detected for two adjacent marker pairs located on the long arm of chromosome 3, but not for marker pairs in other genomic regions. Our results suggest that heterozygosity influences recombination in plant breeding, and cannot be ignored in genetic mapping of genomes.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

美味猕猴桃原生质体再生植株细胞遗传学研究 Ⅰ．体细胞染色体数目的变化

研究了美味猕猴桃叶愈伤组织原生质体再生植株和母株（ＡｃｔｉｎｉｄｉａｄｅｌｉｃｉｏｓａｌｉｎｅＮｏ．２６）茎尖体细胞染色体数目。结果表明：母株２ｎ＝６ｘ＝１７４。所测２９株再生植株的茎尖体细胞染色体数目差异显著,多为非整倍体类型,占所测植株的７２．４％左右;体细胞染色体数目介于１４２－３１０条之间,其中２ｎ＝６ｘ＝１７４约占２０．７％,少于１７４条染色体的植株约占３１．０％,超过１７４条染色体的植株则占４８．３％左右。个别单株部分茎尖体细胞在有丝分裂后期出现染色体桥、断片和落后染色体等异常现象。并对以上现象进行了扼要的讨论。     

Site-specific cleavage of chromosomes in vitro through Cre-lox recombination.

Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences. Site-specific cleavage of lox sites in the tobacco genome was also demonstrated. This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo. Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

Comparative linkage map of the Solanum lycopersicoides and S. sitiens genomes and their differentiation from tomato.

The wild nightshades Solanum lycopersicoides and Solanum sitiens are closely affiliated with the tomatoes (Lycopersicon spp.). Intergeneric hybridization with cultivated tomato (Lycopersicon esculentum) is impeded by strong reproductive barriers including hybrid sterility and suppressed recombination. Conservation of genome structure between these nightshades and tomato was studied by construction of a genetic map from F2 S. sitiens x S. lycopersicoides and comparison with existing maps of tomato. Owing to self-incompatibility of the F1, two hybrid plants were crossed to obtain a population of 82 F2 individuals. Using 166 previously mapped RFLP markers and 5 restriction enzymes, 101 loci polymorphic in the S. sitiens x S. lycopersicoides population were identified. Analysis of linkage between the markers resulted in a map with 12 linkage groups covering 1192 cM and one unlinked marker. Recombination rates were similar to those observed in tomato; however, significant segregation distortion was observed for markers on 7 out of the 12 chromosomes. All chromosomes were colinear with the tomato map, except for chromosome 10, where a paracentric inversion on the long arm was detected. In this region, S. sitiens and S. lycopersicoides share the same chromosomal configuration previously reported for potato (S. tuberosum) and pepper (Capsicum), suggesting that of tomato is derived. The 10L inversion explains the lack of recombination detected among homeologous chromosomes of intergeneric hybrids in this region. On this basis, we recognize two principle genomes, designated L for the Lycopersicon spp., and S for S. lycopersicoides and S. sitiens, the first examples of structural differentiation between tomato and its cross-compatible wild relatives.     

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.     

“Arabidobrassica”: Chromosomal recombination and morphogenesis in asymmetric intergeneric hybrid cells

A somatic hybrid cell line, cloned from an individual protoplast-fusion product between Arabidopsis thaliana and Brassica campestris, gave rise to formation of numerous plants differing drastically in morphology. Analysis of these various regenerants, all of which originated from one and the same heterokaryon derived from the fusion of two cells, shows the unspecific elimination of chromosomes of both parental species during the callus growth phase. Whereas the parental cells have so far not been sucessfully regenerated into plants, several of their different asymmetric hybrids are capable of morphogenesis. Furthermore, chromosomal analysis indicates extensive recombination. Most of the plants are predoinantly morphologically regular. Abnormalities are mostly limited to the flowers which tend to undergo phyllody. The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable. The transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.     

Epigenetic control may explain large within-plant heterogeneity of meiotic behavior in telocentric trisomics of rye

In telocentric trisomics (telotrisomics) of organisms in which the chromosomes normally have two distinct arms, a single chromosome arm with a centromere is present in addition to a complete diploid set of chromosomes. It is the simplest form of polysomy and suitable for analyzing meiotic pairing and recombination patterns in situations where chromosomes compete for pairing. When no suitable meiotic chromosome markers are available, four metaphase I configurations can be distinguished. Their relative frequencies are indicative of the pairing and recombination patterns. In short arm (1RS) telotrisomics of chromosome 1R of rye (Secale cereale) we observed great differences in pairing and recombination patterns among spikes from different tillers and clones of the same plants. Anthers within spikes were only very rarely different. We analyzed a large number of genotypes, including inbreds as well as hybrids. The effects of genetic and environmental conditions on heterogeneity, if any, were limited. Considering that the reproductive tissue of a spike is derived from one primordial cell, it seems that at the start of sexual differentiation there was variation among cells in chromosomal control, which at meiosis determines pairing and crossing-over competence. We suggest that it is an epigenetic system that rigidly maintains this pattern through generative differentiation. In competitive situations the combination most competent for pairing will pair preferentially, forming specific meiotic configurations with different frequencies for different spikes of the same plant. This would explain the heterogeneity between spikes and the homogeneity within spikes. The epigenetic system could involve chromatin conformation or DNA methylation. There were no signs of heterochromatinization.     

The tobacco A-type cyclin, Nicta;CYCA3;2, at the nexus of cell division and differentiation

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly from animals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in animals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. We previously identified the tobacco A-type cyclin Nicta;CYCA3;2 as an early G1/S-activated gene. Here, we show that antisense expression of Nicta;CYCA3;2 in tobacco plants induces defects in embryo formation and impairs callus formation from leaf explants. The green fluorescent protein (GFP)-Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm. Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation, whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plants that overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modified morphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and root regeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in the control of plant cell division and differentiation.     

A novel Ti-plasmid-convertible λ phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants

A new λ phage vector system, λTI, has been constructed to facilitate genetic complementation of higher plant mutations. The λTI vectors are stable, and by using the Cre— lox site-specific recombination, are automatically convertible into Ti-plasmid binary vectors which are capable of expressing genes in higher plants. Two λTI vectors were constructed: (i) λTI1, which can generate a Ti-plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) λTI2, which can generate a Ti-plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these λTI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation-competent clones.     

Nuclear gametophytic genes from chromosome arm 1RS improve regeneration of wheat microspore-derived embryos.

The effect of the 1RS chromosome arm from rye on plant regeneration from microspore-derived embryos was studied using anther culture technology with genotypes carrying the 1BL-1RS translocated chromosomes, the normal wheat chromosome 1BL-1BS, and ditelosomic lines DT 1BS and DT 1BL. A significant difference was observed in microspore-derived green plants between chromosome structure concerned with 1RS and 1BS arms. An analysis of the inheritance of the 1B-1R translocation was performed on the basis of the frequency of male gametes 1BL-1RS in the microspore-derived green plants and that of the 1B-1R translocation inherited through the pollen or the egg cell from structurally heterozygous hybrids 1BL-1BS/1BL-1RS. Both the normal 1B and the translocated 1BL-1RS chromosomes were sexually transmitted through the pollen grains with the same frequency. The 1BL-1RS chromosome is only transmitted through 45% of the egg cells. On the contrary, two-thirds of the microspore-derived green plants regenerated from the anther culture experiments possess the translocated chromosome. The involvement of the rye chromosome arm 1RS from 'Aurora' on regeneration capacity of the microspore-derived embryos has been proposed through the effect of a "gametophytic gene."     

Genetic and physical mapping of sequence-specific amplified polymorphic (SSAP) markers on the 1RS chromosome arm of rye in a wheat background

Three rye-specific repeated sequences, pSc10C, pSc20H and R173-1, were used to design sequence-specific anchored primers. These primers and 16 restriction site-specific adaptor primers were used in all possible combinations to establish sequence-specific amplified polymorphic (SSAP) markers for the 1RS chromosome arm of rye in a wheat background. Thirty 1RS-specific SSAP markers were detected in 19 primer combinations. Along with six markers localised previously on 1RS, 26 of the SSAP markers were mapped genetically in wheat genotypes carrying recombinant 1BL.1RS translocations. A clear decrease in recombination frequency from distal to proximal regions was observed. Wheat-rye addition lines for the 1R chromosome with different-sized deletions of the short arm were used to physically localise these markers. Physical mapping suggested an even distribution of the SSAP markers along the total length of the 1RS chromosome arm.Communicated by J.W. Snape     

FtsK-dependent and -independent pathways of Xer site-specific recombination.

Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division. Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers. Two recombinases, XerC and XerD, act at the E. coli chromosomal recombination site, dif, and at related sites in plasmids. We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors. We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif. The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers. Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates.