Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Analysis of karyotype and C-banding pattern ofNicotiana plumbaginifolia using two techniques

The morphological study of the chromosomes ofNicotiana plumbaginifolia was carried out using two karyological techniques referred to as the squash method and the protoplast method. The latter involves isolation of protoplasts from unfixed root meristems and provides mitotic images in which the chromosomes, conveniently dispersed and disposed on the same plane, are significantly longer, and better structured, than those obtained by the squash method. A critical analysis of this technique is carried out. The results of this study led us to propose a karyotype forNicotiana plumbaginifolia and to characterize each chromosome by mere morphological examination. This characterization was further detailed by C-banding.     

Plant tumor reversal associated with the loss of marker chromosomes in tobacco cells

Summary The determination of partial genotype, B/b and D/d, for coat colour in dogs, from phenotypic observations, is discussed. It is shown that the probability of a given genotype can be reliably determined where multiple observations on the mating of a given dog are available.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

Denombrement et analyse morphologique des chromosomes chez quelques clones issus d'une souche anergiee de tabac: Relation avec l'anergie,le pouvoir tumoral et l'organogenese

Counting and morphological analysis of chromosomes in some clones derived from habituated tobacco callus cultures: relationship with habituation, tumorous properties and organogenesis     

Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

Field estimates of fitness costs of the pace‐of‐life in an endangered damselfly

Theory predicts that within‐population differences in the pace‐of‐life can lead to cohort splitting and produce marked intraspecific variation in body size. Although many studies showed that body size is positively correlated with fitness, many argue that selection for the larger body is counterbalanced by opposing physiological and ecological selective mechanisms that favour smaller body. When a population split into cohorts with different paces of life (slow or fast cohort), one would expect to detect the fitness–size relationship among and within cohorts, that is, (a) slower‐developing cohort has larger body size and higher fitness than faster‐developing cohort, and (b) larger individuals within each cohort show higher fitness than smaller individuals. Here, we test these hypotheses in capture–mark–recapture field surveys that assess body size, lifespan, survival and lifetime mating success in two consecutive generations of a partially bivoltine aquatic insect, Coenagrion mercuriale, where the spring cohort is slower‐developing than the autumn cohort. As expected, body size was larger in the slow‐developing cohort, which is consistent with the temperature‐size rule and also with the duration of development. Body size seasonal variation was greater in slow‐developing cohort most likely because of the higher variation in age at maturity. Concordant with theory, survival probability, lifespan and lifetime mating success were higher in the slow‐developing cohort. Moreover, individual body size was positively correlated with survival and mating success in both cohorts. Our study confirms the fitness costs of fast pace‐of‐life and the benefits of larger body size to adult fitness.     

Frameshift deletion mechanisms in Egyptian Duchenne and Becker muscular dystrophy families

Partial gene deletion is the major type of mutation leading to Duchenne muscular dystrophy (DMD) and its mild allelic form, Becker muscular dystrophy (BMD). Amplification of the genomic DNAs of 152 unrelated dystrophin patients using multiple primers detected 78 (51.3%) probands with deletion mutations. We predicted the translational reading frame for all the deletions in Egyptian dystrophin males. The frameshift rule was confirmed positively ranging for 50 to 67% of the cases depending on the type of disease. We discuss ways of accounting for some exceptions from the frameshift hypothesis in the central and proximal regions. These explanations may help in developing procedures for reducing the severity of dystrophin phenotypes to restore the correct frame by disrupting the translational fidelity. Great efforts have been put into the development of effective 'gene correction' procedures via such intrinsic mechanisms. In addition, we mapped regional difference in deletion mutation frequencies within the DMD gene locus between the different Egyptian governorates. There were no double deletions in the Egyptian dystrophin males.