Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

A stochastic model of recombination during conjugation in Escherichia coli K-12.

A mathematical model is constructed in which recombination in E. coli K-12 is considered as a stochastic Markov process. The model takes into account the possibility of inclusion in the recombinant structure of the origin of the donor chromosome and makes it possible to correctly describe results of irradiation of the donor Hfr before crossing. Formulae are deduced for frequencies of unselected markers for cases when the counter-selected maternal marker lies in the distal or proximal region of the chromosome.     

The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.     

Conjugational chromosome transfer—Complete or partial? Kinetics of chromosome transfer in bacterial conjugation

A mathematical model is proposed which describes the kinetics of chromosome transfer during conjugation of bacteria Escherichia coli K-12. The kinetics of pairing and the appearance of individual recombinants is expressed quantitatively, and on this basis the quantity of donor genetic material transferred to the recipient cell during crossing is considered. Predictions of the theoretical model are compared with experiments on transfer of radioactive label. This comparison indicates that the whole donor chromosome is transferred during conjugation.     

Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu.

Conjugational gene transfer was established in Erwinia carotovora subsp. carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients. It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca. 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold. Using this system it was possible to order 17 chromosomal mutations. The behavior of Mu in E. carotovora subsp. carotovora was also studied.     

The construction of a substitution library of recombinant backcross lines in Brassica oleracea for the precision mapping of quantitative trait loci.

The currently available methods for locating quantitative trait loci (QTLs) and measuring their effects in segregating populations lack precision unless individual QTLs have very high heritabilities. The use of recombinant backcross lines containing short regions of donor chromosome introgressed into a constant recipient background permits QTLs to be located with greater precision. The present paper describes the use of molecular markers to introgress defined short regions of chromosome from a donor doubled haploid calabrese line of Brassica oleracea (var. italica) into a recipient short generation variety (Brassica oleracea var. alboglabra). We demonstrate that in just two or three generations of backcrossing, combined with selection for mapped molecular markers, the generation of a library of recombinant backcross lines is feasible. The possible use and refinement of these lines are discussed. Key words : backcrossing, Brassica oleracea, introgression, molecular markers, near-isogenic lines, QTL mapping, recombinant backcross lines, substitution lines.     

Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.     

Genetic mapping of the obligate methylotroph Methylobacillus flagellatum: characteristics of prime plasmids and mapping of the chromosome in time-of-entry units.

A pULB113 (RP4::mini-Mu cts) plasmid was used to generate a library of prime plasmids carrying fragments of the Methylobacillus flagellatum genome. The genes carried by these prime plasmids were identified by complementation after transfer to suitably marked Escherichia coli and Pseudomonas aeruginosa strains. The hybrid plasmids were used for complementation mapping with a range of E. coli, M. flagellatum, and P. aeruginosa mutants. A preliminary map of the M. flagellatum genome section with seven groups of linked markers was obtained. Three of seven groups contain an overlapping sequence of cloned genes and can be considered as one large group of linked genes. A high-frequency-of-recombination donor of M. flagellatum (strain MFK64) mobilized the chromosome in a polarized manner from a single transfer origin. The donor was used to construct a time-of-entry map of the M. flagellatum chromosome. This was achieved by determining the time of entry of six randomly dispersed markers, four of which are included in known groups of linked markers. The linear map of M. flagellatum reported here consists of 44 markers.     

Microsatellites confirm the authenticity of inter-varietal chromosome substitution lines of wheat (Triticum aestivum L.)

Ninety-five wheat microsatellite markers (WMS) were used to verify the authenticity of the set of Saratovskaya 29/Yanetzkis Probat inter-varietal wheat chromosome substitution lines developed using Saratovskaya 29 as the recipient variety. Polymorphic markers were available for all chromosome arms except 4DS, 6DS and 7DS. Each chromosome substitution line was tested by 2–8 microsatellite markers. The results demonstrate that most of the lines are correct. Out of 21 lines tested 17 showed the expected microsatellite pattern of the donor variety. Two entire chromosomes, 1B and 7A, and two chromosome arms, 3AL and 6DS, were not substituted with Yanetzkis Probat in their respective lines. Three microsatellite markers located in the distal regions of chromosome arms 4AL, 3BS and 5BL in the corresponding substitution lines did not reveal the expected microsatellite pattern of the recipient variety. The possible causes of the incorrect substitution line development and the appearance of incorrect distal microsatellite markers are discussed. The data confirm the idea that microsatellite markers provide ideal tools for testing the authenticity of genetic stocks of wheat. Received: 27 August 1999 / Accepted: 8 October 1999     

Chromosome Mapping of Pasteurella pseudotuberculosis by Interrupted Mating

Pasteurella pseudotuberculosis, containing the Escherichia coli plasmid F'lac, transferred its chromosome in an oriented manner to each of five multiply auxotrophic strains of P. pseudotuberculosis. In a mating system containing gelatin, glucose, and phosphate buffer, a maximum of 0.02% of the donor cells transferred lead markers. The donor population was counterselected with nalidixic acid. We established the entry time of seven markers as follows: proline (11 min); arginine (14 min); histidine (14 min); threonine (25 min); lysine (50 min); tyrosine (67 min); and tryptophan (77 min). However, an analysis of the inheritance of unselected markers did not support the simplest assumption that the chromosome was transferred as Origin... pro... arg his... thr... lys... tyr... trp.... The markers common to all five recipients, arg and his, were closely linked, but of the five other markers, each unique to a different recipient strain, only trp was linked to arg and his. Our data suggest that the Pasteurella chromosome is transferred in more than one linkage group.     

Size of donor chromosome segments around introgressed loci and reduction of linkage drag in marker-assisted backcross programs.

This article investigates the efficiency of marker-assisted selection in reducing the length of the donor chromosome segment retained around a locus held heterozygous by backcrossing. First, the efficiency of marker-assisted selection is evaluated from the length of the donor segment in backcrossed individuals that are (double) recombinants for two markers flanking the introgressed gene on each side. Analytical expressions for the probability density function, the mean, and the variance of this length are given for any number of backcross generations, as well as numerical applications. For a given marker distance, the number of backcross generations performed has little impact on the reduction of donor segment length, except for distant markers. In practical situations, the most important parameter is the distance between the introgressed gene and the flanking markers, which should be chosen to be as closely linked as possible to the introgressed gene. Second, the minimal population sizes required to obtain double recombinants for such closely linked markers are computed and optimized in the context of a multigeneration backcross program. The results indicate that it is generally more profitable to allow for three or more successive backcross generations rather than to favor recombinations in early generations.     

Altered linkage values in phage P22-mediated transduction caused by distant deletions or insertions in donor chromosomes

Summary The effects of distant deletions or insertions in the Salmonella typhimurium donor strains on P22-mediated cotransducibility of genetic markers was studied. We found that deletions of histidine operon, unit 44 of the chromosome map, changed the linkage of markers purF and aroC (unit 49) and pyrF and trpA (unit 34). They did not change the linkage of more distant markers pyrE and cysE. The effect of three types of insertions was examined. The donor strains carried F factor, Tn10 transposon or pi-his duplication inserted close to histidine operon. These insertions caused alteration of purF-aroC linkage while pyrF-trpA cotransduction values were not affected. These data show that the effect of the chromosome rearrangements extends to at least 5% of S. typhimurium chromosome length and may reach as much as 10% of it. Our results are in agreement with the model of Chelala and Margolin (1974) concerning formation of transduction particles. They indicate that the cotransducibility changes caused by deletions or insertions extent further than it might have been expected from previous reports.     

Frequency and fidelity of alien chromosome transmission in Gossypium hexaploid bridging populations.

The Australian diploid Gossypium species possess traits of potential agronomical value, such as gossypol-free seeds and Fusarium wilt resistance. However, they belong to the tertiary germplasm pool, which is the most difficult group of species from which to introgress genes into G. barbadense L. and G. hirsutum L. Interspecific triploid hybrids can be generated but they are sterile. The sterility barrier can be overcome using synthetic polyploids as introgression bridges, but whether there is sufficient homoeologous chromosome interaction at meiosis to allow recombination is still an open question. To ascertain, genetically, observable levels of homoeologous introgression, 2 synthetic hexaploid lines (2x G. hirsutum x G. australe and 2x G. hirsutum x G. sturtianum) were crossed to G. hirsutum to generate pentaploid F1 plants that, in turn, were backcrossed to G. hirsutum to generate BC1 and BC2 multiple alien chromosome addition lines (MACALs). Gossypium australe F. Muell. and G. sturtianum Willis chromosome-specific markers were used to track the frequency and fidelity of chromosome transmission to the BC1 and BC2 MACALs. The chromosomal location of the AFLP markers was determined by their distribution among the MACALs and confirmed in parental F2 families. Roughly half the available chromosomes were transmitted to the G. hirsutum x G. australe (54%) and G. hirsutum x G. sturtianum (52%) BC1 MACALs. The BC2 MACAL families again inherited about half of the available chromosomes. There were, however, notable exceptions for specific chromosomes. Some chromosomes were preferentially eliminated, while others were preferentially transmitted. Consistent with the genomic stability of Gossypium synthetic polyploids, the de novo loss or gain of AFLP fragments was rarely observed. While restructuring of the donor G. australe and G. sturtianum chromosomes was observed, this is more likely the result of chromatin loss, and no clear cases of introgression of donor chromatin into the recipient G. hirsutum genome were observed.     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

White spotting in the domestic cat (Felis catus) maps near KIT on feline chromosome B1

Five feline-derived microsatellite markers were genotyped in a large pedigree of cats that segregates for ventral white spotting. Both KIT and EDNRB cause similar white spotting phenotypes in other species. Thus, three of the five microsatellite markers chosen were on feline chromosome B1 in close proximity to KIT; the other two markers were on feline chromosome A1 near EDNRB. Pairwise linkage analysis supported linkage of the white spotting with the three chromosome B1 markers but not with the two chromosome A1 markers. This study indicates that KIT, or another gene within the linked region, is a candidate for white spotting in cats. Platelet-derived growth factor alpha (PDGFRA) is also a strong candidate, assuming that the KIT-PDGFRA linkage group, which is conserved in many mammalian species, is also conserved in the cat.     

Linkage analysis of autosomal dominant atrio ventricular canal defects: exclusion of chromosome 21

The association between trisomy 21 and a high incidence of atrioventricular canal defects (AVCDs) indicates that a locus on chromosome 21 is involved in this congenital heart defect. We have investigated whether a genetic locus on chromosome 21 is also involved in familial nonsyndromic AVCDs. Short tandem repeat polymorphisms (STRPs) from chromosome 21 were used for linkage analysis of a family having multiple members affected with AVCDs. In this family, the gene for AVCDs is transmitted as an autosomal dominant with incomplete penetrance. The affected family members are nonsyndromic and have normal karyotypes. Two-point and multipoint linkage analyses produced significantly negative LOD scores for all informative markers. A comparison of the overlapping exclusion distances obtained for each marker at LOD equal -2.0 with the 1000:1 consensus genetic map of the markers, excludes chromosome 21 as the genetic location for AVCDs in this family. The exclusion of chromosome 21 indicates that another gene, not located on chromosome 21, is involved in atrioventricular canal defect formation.     

Fertility factors in Pseudomonas putida: selection and properties of high-frequency transfer and chromosome donors.

The octane plasmid (OCT) in Pseudomonas putida strains has been shown to be transferred at low frequency. However, bacteria which had newly received this plasmid showed a transient increase in donor ability. Using Octane+ P. putida as the donor, the transfer of most chromosomal markers was shown to be independent of OCT transfer, whereas the mobilization of the octanoate catabolism genes (octanoic and acetate) was dependent on OCT plasmid transfer. The presence of a fertility factor termed FPo has been postulated to explain these results. Strains carrying only this fertility factor have been obtained from strains carrying both OCT and FPo plasmids. Strains in which the OCT plasmid was transferred at high frequencies have also been isolated, and chromosome mobilization by OCT and FPo has been compared. A different gradient of transmission by OCT and FPo has been observed. It has also been shown that chromosome transfer by OCT was dependent on the bacterial recombination system, whereas the chromosome transfer by FPo was unaffected by the presence of a rec mutation in the donor strain.     

Molecular-genetic maps for group 1 chromosomes of Triticeae species and their relation to chromosomes in rice and oat

Group 1 chromosomes of the Triticeae tribe have been studied extensively because many important genes have been assigned to them. In this paper, chromosome 1 linkage maps of Triticum aestivum, T. tauschii, and T. monococcum are compared with existing barley and rye maps to develop a consensus map for Triticeae species and thus facilitate the mapping of agronomic genes in this tribe. The consensus map that was developed consists of 14 agronomically important genes, 17 DNA markers that were derived from known-function clones, and 76 DNA markers derived from anonymous clones. There are 12 inconsistencies in the order of markers among seven wheat, four barley, and two rye maps. A comparison of the Triticeae group 1 chromosome consensus map with linkage maps of homoeologous chromosomes in rice indicates that the linkage maps for the long arm and the proximal portion of the short arm of group 1 chromosomes are conserved among these species. Similarly, gene order is conserved between Triticeae chromosome 1 and its homoeologous chromosome in oat. The location of the centromere in rice and oat chromosomes is estimated from its position in homoeologous group 1 chromosomes of Triticeae.