Transmission and recombination of extranuclear genes during sexual crosses in Aspergillus nidulans

Summary Three extranuclear mitochondrial mutations in Aspergillus nidulans, (oliA1), (camA1) and (cs67), were used as markers in sexual crosses to provide information on the frequencies of transmission and recombination of the mitochondrial genome. Any individual perithecium contained ascospores of only one extranuclear genotype.Using mono-, bi- and trifactorial crosses it was found that all three markers could be recovered from the progeny, although the transmission frequencies were different for each marker. This bias was present irrespective of the nuclear background or the presence of selective agents in the medium on which the cross was established. These findings enable a series of transmission strength to be established, as shown below:- (cs67,{\text{ }}camA1) > ( + ) = (cs67) > (oliA1,cs67) \hfill \\ {\text{ }} > (oliA1) > (oliA1,{\text{ }}camA1) \hfill \\ \end{gathered} $$ " align="middle" border="0"> However, the numbers of recombinants isolated were so variable as to make this form of analysis unsuitable for mapping the mitochondrial genome.     

Mobilization of plasmid-borne drug resistance determinants for transfer from Pseudomonas aeruginosa to Escherichia coli.

Summary Two- and three-point extranuclear crosses have been carried out via heterokaryons involving the three extranuclear mitochondrial markers of Aspergillus nidulans: (oliA1), (cs67) and (camA112). All three markers appear to be located on a single functional mitochondrial genome. Recombination between all three pairs of extranuclear markers appears to be equally frequent, suggesting a lack of genetic linkage. An important feature of these results is the variable and often marked non-equality of frequency of reciprocal classes of recombinants.     

Extranuclear recombination in Aspergillus nidulans: closely-linked multiple chloramphenicol- and oligomycin-resistance loci.

Summary A nuclear, chloramphenicol-sensitive mutant cas-1 has been isolated which is cross sensitive to a number of drugs, including oligomycin and cycloheximide. Approximately one-third of the chloramphenicol-resistant mutants isolated from mutagenized conidia of this strain were found to be extranuclear, and exhibited a variety of phenotypes. One of these mutants, designated (camB51), was slow growing on drug-free medium and recombined at low frequency with the previously described mutant (camA112) (Gunatilleke et al., 1975).The majority of extranuclear oligomycin-resistant mutants isolated from cas-1 were indistinguishable from (oliA1) (Rowlands and Turner, 1973). Two mutants, (oliB322) and (oliB332), with similar but not identical phenotypes to (oliA1), recombined with the latter at low frequency but not with each other, thus representing a new class of extranuclear mutants.     

Nuclear and mitochondrial suppression of a mitochondrially inherited cold-sensitive mutation in Aspergillus nidulans

Partial suppressors of a mitochondrially inherited mutation, [cs-67], conferring cold-sensitivity at 20 degrees C were identified. These mapped at one mitochondrial and four unlinked nuclear loci. Most suppressors partially restored the cytochrome aa3 deficiency of the cold-sensitive strain at 20 degrees C. Strains carrying two or more suppressors and [cs-67] showed considerably impaired growth. This effect was temperature-dependent, being more severe at 37 degrees C, and was not expressed in the presence of the [cs-67+] allele. The cytochrome oxidase activity of one of these strains was no more heat-sensitive than that of the wild-type implying that these mutations did not directly modify cytochrome oxidase. The wild-type strain grown in the presence of chloramphenicol and the cold-sensitive strain grown at 20 degrees C had similar cytochrome spectra and mitochondrial membrane protein profiles on sodium dodecyl sulphate gradient acrylamide gels. [cs-67] conferred pleiotropically a low level of resistance to paramomycin at 37 degrees C. It is suggested that [cs-67] and the suppressors act at the level of the mitochondrial ribosome.     

Recombination between the extranuclear genes conferring oligomycin resistance and cold sensitivity in Aspergillus nidulans

Summary Recombination has been demonstrated between the extranuclear loci (oliA1) and (cs67) of Aspergillus nidulans. The stability of the double mutant recombinant and the fact that it formed smaller colonies than either parent at the non-permissive temperature are strong evidence that physical recombination of the extranuclear DNA has occurred rather than simple mixing. A method has been developed for quantifying the extranuclear recombination frequency, thus providing a means of mapping the A. nidulans mitochondrial genome. The data obtained suggests that the two loci are not closely linked.     

Mitochondrial Genetics. Xii. an Oligomycin-Resistant Mutant Localized at a New Mitochondrial Locus in SACCHAROMYCES CEREVISIAE

Three antibiotic-resistance mutations were isolated from strain FL496–2B: two are independent Mendelian genes, one conferring both oligomycin and venturicidin resistance (oli^R₄₉₆) and the other conferring cycloheximide resistance (cyh^R₄₉₆). The third is a mitochondrial mutation, O^R₉, and confers a low level of oligomycin resistance to cells (in vivo) but not to the extracted mitochondrial ATPase (in vitro). This mutation is located on the mitochondrial DNA at a new locus [OLI4] linked to [OLI2] and independent from [OLI1] and [OLI3] and from the other mitochondrial loci.

All three mutations (O ^R₉, oli^R₄₉₆, cyh^R₄₉₆ ) were found without any selection, in the same prototrophic haploid strain, which contained unknown resistances to antibiotics.

Some physiological, genetical and biochemical properties of the mitochondrial mutation are described.

     

Further Evidence for a Polymorphism in Gametic Segregation in the Tetraploid Treefrog HYLA VERSICOLOR Using a Glutamate Oxaloacetic Transaminase Locus

Intra- and interspecific cross combinations between the tetraploid treefrog Hyla versicolor, and between H. versicolor and the diploid treefrog Hyla chrysoscelis were performed. Progeny phenotypes resulting from these crosses were examined electrophoretically using a polymorphic glutamate oxaloacetic transminase (GOT-1) locus, to determine the mechanism of chromosome segregation in H. versicolor, and to test theoretical expectations for isozyme expression in interspecific (2n x 4n or 4n x 2n) hybrids. In some intraspecific tetraploid crosses progeny phenotypes fit a disomic mode of segregation, whereas in other crosses a tetrasomic mode of segregation was the most probable. Additional crosses produced phenotypic ratios that conformed to either a disomic or tetrasomic mode of segregation. These results suggest that a polymorphism, with respect to segregation of gametes, exists in H. versicolor, resulting from differences in chromosome pairings during meiosis I. This polymorphism in gametic segregation occurred in both sexes. Certain crosses, however, produced phenotypic ratios that did not conform to any chromosome segregation model. Progeny phenotypes observed from most interspecific crosses conformed to expected interspecific isozyme staining intensity models. Symmetrical heterozygotes, representing either a single dose for both alternate alleles or double doses for both alternate alleles, were also observed. Such phenotypes are unexpected in triploid progeny. A null allele was postulated to account for the aberrant segregation ratios and phenotypes observed in certain intra- and interspecific crosses.     

Two Cell Division Cycle Mutants of SACCHAROMYCES CEREVISIAE Are Defective in Transmission of Mitochondria to Zygotes

Mutations in CDC genes of S. cerevisiae disrupt the cell cycle at specific stages. The experiments reported here demonstrate that two CDC genes, CDC5 and CDC27, are necessary for mitochondrial segregation as well as for nuclear division. The defect in the transmission of mitochondria was revealed by the examination of uninucleate and binucleate progeny of transient heterokaryons generated by using the kar1-1 mutation that disrupts nuclear fusion. One of the parents lacked mitochondrial DNA (ρ⁰) whereas the other parent had functional mitochondria (ρ⁺). When the parents of the heterokaryon were both wild-type (CDC), nearly all progeny received mitochondria at 21° and at 34°. Thirty-four of the 36 cdc mutations tested had no defect in transmission of mitochondria to zygotic progeny in crosses in which one parent was a cdc mutant and the other parent was not (CDC). However, the cdc5 and cdc27 mutations prevented the transmission of mitochondria to cdc progeny at 34° but not at 21°; CDC progeny received mitochondria at either temperature. This defect was observed in crosses of cdc5 or cdc27 by wild-type cells regardless of which parent donated mitochondria to the zygote. The defect in mitochondrial transmission cosegregated in meiotic tetrads with the defect in mitosis demonstrating that both are likely to be caused by the same temperature-sensitive mutation. These results indicate that the CDC5 and CDC27 gene products are essential in two motility-related processes: mitochondrial movement from the zygote to the progeny and in mitosis.—Furthermore, the results suggest that the function performed by the CDC5 and CDC27 gene products for mitochondrial transmission differ in some fundamental way from the function performed for mitosis. The function necessary for mitosis can be supplied to the cdc5 (or cdc27) nucleus by the CDC5 (or CDC27) nucleus in the same heterokaryon but the function necessary for mitochondrial transmission cannot. Perhaps the function needed for mitochondrial transmission must be performed in the cell cycle preceding the actual segregation of mitochondria whereas the function needed for nuclear segregation can be performed at the time that mitosis occurs.     

On the formation of rho? petites in yeast

Summary The inheritance of an extrakaryotic mutation conferring temperature-sensitive growth on nonfermentable substrates and a high frequency of mutation to rho^– has been studied. Multifactorial crosses (rho⁺xrho⁺) involving this mutation T ₈ ^S and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin or paromomycin revealed: a) Mutation T ₈ ^S is localized on the mitDNA, referring to a new gene locus TSM1. b) Locus TSM1 appears to be weakly linked to the locus PAR1 and to the loci RIB1 and RIB3 but unlinked to the locus OLI1. c) The position of TSM1 is between PAR1 and the two closely linked loci RIB1 and RIB3, OLI1 is outside and not linked to the segment PAR-TSM-RIB. d) Mutation T ₈ ^S does not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus .     

Intracellular Population Genetics: Evidence for Random Drift of Mitochondrial Allele Frequencies in SACCHAROMYCES CEREVISIAE and SCHIZOSACCHAROMYCES POMBE

We report evidence for random drift of mitochondrial allele frequencies in zygote clones of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Monofactorial and bifactorial crosses were done, using strains resistant or sensitive to erythromycin (alleles E^R, E^S), oligomycin (O^R, O^S), or diuron (D^R, D^S). The frequencies of resistant and sensitive cells (and thus the frequencies of the resistant and sensitive alleles) were determined for each of a number of clones of diploid cells arising from individual zygotes. Allele frequencies were extremely variable among these zygote clones; some clones were "uniparental," with mitochondrial alleles from only one parent present. These observations suggest random drift of the allele frequencies in the population of mitochondrial genes within an individual zygote and its diploid progeny. Drift would cease when all the cells in a clone become homoplasmic, due to segregation of the mitochondrial genomes during vegetative cell divisions. To test this, we delayed cell division (and hence segregation) for varying times by starving zygotes in order to give drift more time to operate. As predicted, delaying cell division resulted in an increase in the variance of allele frequencies among the zygote clones and an increase in the proportion of uniparental zygote clones. The changes in form of the allele frequency distributions resembled those seen during random drift in finite Mendelian populations. In bifactorial crosses, genotypes as well as individual alleles were fixed or lost in some zygote clones. However, the mean recombination frequency for a large number of clones did not increase when cell division was delayed. Several possible molecular mechanisms for intracellular random drift are discussed.     

Phenotypic Expression of rkn1-Mediated Meloidogyne incognita Resistance in Gossypium hirsutum Populations

The root-knot nematode Meloidogyne incognita is a damaging pest of cotton (Gossypium hirsutum) worldwide. A major gene (rkn1) conferring resistance to M. incognita was previously identified on linkage group A03 in G. hirsutum cv. Acala NemX. To determine the patterns of segregation and phenotypic expression of rkn1, F₁, F₂, F_2:3, BC₁F₁ and F_2:7 recombinant inbred lines (RIL) from intraspecific crosses between Acala NemX and a closely related susceptible cultivar Acala SJ-2 were inoculated in greenhouse tests with M. incognita race 3. The resistance phenotype was determined by the extent of nematode-induced root galling and nematode egg production on roots. Suppression of root galling and egg production was highly correlated among individuals in all tests. Root galling and egg production on heterozygous plants did not differ from the susceptible parent phenotype 125 d or more after inoculation, but were slightly suppressed with shorter screening (60 d), indicating that rkn1 behaved as a recessive gene or an incompletely recessive gene, depending on the screening condition. In the RIL, rkn1 segregated in an expected 1 resistant: 1 susceptible ratio for a major resistance gene. However, within the resistant class, 21 out of 34 RIL were more resistant than the resistant parent Acala NemX, indicating transgressive segregation. These results suggest that rkn1-based resistance in G. hirsutum can be enhanced in progenies of crosses with susceptible genotypes. Allelism tests and molecular genetic analysis are needed to determine the relationship of rkn1 to other M. incognita resistance sources in cotton.     

The use of genetic markers selected in vitro for the isolation and genetic verification of intraspecific somatic hybrids of Nicotiana tabacum L.

Summary A scheme employing genetic markers obtained by in vitro selection was developed for the stringent isolation of hybrid somatic cells of Nicotiana tabacum. Mesophyll protoplasts that carried two dominant alleles of nuclear genes conferring resistance to the herbicide picloram (pmR1) and the ability to utilize glycerol as the sole source of carbon (Gut) were fused with suspension-culture protoplasts that were marked with the dominant nuclear allele (HuR9) conferring resistance to hydroxyurea. Putative somatic hybrid cell lines were identified by selecting for the Gut and HuR9 markers, followed by an assay for the unselected marker PmR1. Plants regenerated from six of these cell lines were proved to be true somatic hybrids by demonstrating the segregation of each of the three parental markers in the progeny of crosses of those plants with normal seed-derived plants.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.     

Evidence for a Polymorphism in Gametic Segregation Using a Malate Dehydrogenase Locus in the Tetraploid Treefrog HYLA VERSICOLOR

Artificial cross combinations of tetraploid Hyla versicolor were analyzed electrophoretically using a polymorphic malate dehydrogenase locus (MDH-1) to determine the mechanism of chromosome segregation. Models for differentiating between disomic and tetrasomic inheritance are presented and tested. In some crosses progeny genotypes fit a disomic mode of segregation. In other crosses there is only evidence for a tetrasomic mode of segregation. Additional crosses produced genotypic ratios which conformed to either a disomic or tetrasomic mode of segregation. The same type of inheritance was demonstrated for any individual when used in multiple cross combinations. These results suggest that there exists in H. versicolor a polymorphism with respect to segregation of gametes, resulting from differences in chromosome pairings during meiosis I.     

Mitochondrial oligomycin-resistance mutations affecting the proteolipid subunit of the mitochondrial adenosine triphosphatase.

Two independently isolated oligomycin resistant mutants of Saccharomyces cerevisiae have been studied. The oligomycin resistance is conferred in each case by a single mutation at an oliA locus. In both strains the proteolipid subunit of the mitochondrial ATPase (subunit 9) shows an apparent increase in molecular weight as judged by its mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Variable effects are seen on other subunits. These results suggest that oliA loci may play some role in the determination of proteolipid ATPase subunit.     

Nuclear-extranuclear interactions affecting oligomycin resistance in Aspergillus nidulans.

Summary The extranuclear mitochondrial oligomycin-resistant mutation ofAspergillus nidulans, (oliA1), was transferred asexually into four nuclear oligomycin-resistant strains of different phenotypes. In all four cases, the possession of the nuclear plus extranuclear mutation led to an increase in the in vivo level of oligomycin resistance. In two cases, the altered cytochrome spectrum and impaired growth ability determined by (oliA1) were suppressed by the nuclear mutations. In the third case, the in vitro oligomycin resistance of the double mutant ATPase was dramatically increased above that of either of the component single mutant strains, indicating a synergystic interaction between the nuclear and extranuclear gene products. In the fourth case, the double mutant became cold-sensitive.A new extranuclear mitochondrial oligomycin-resistant mutation (oliB332) is described. This mutant is phenotypically similar to, though not identical with, (oliA1) but is separable by recombination.A range of nuclear oligomycin-resistant mutants have been mapped. Despite presenting five distinctly different phenotypes, they all map at the same locus.     

Extrachromosomal inheritance in Schizosaccharomyces pombe

Summary Primary and secondary spore clones were analyzed from two- and three-factor crosses involving the mitochondrial markers conferring resistance to antimycin (A ^R ), chloramphenicol (C ^R ), and erythromycin (E ^R ). As in zygote clones (Seitz-Mayr et al., 1978), transmission of markers is higher in two-factor trans-crosses than in cis-crosses. Except transmission of C ^R in the cross A ^R C ^R E ^R xA ^S C ^{S E S} , no significant differences between cis- and trans-configuration were observed in three-factor crosses. In contrast to zygote clones, in spore clones transmission rates of the two or three markers in a given cross are roughly equal. 18 out of 20 secondary spore clones of different mitochondrial phenotypes appeared to be homoplasmic, whereas 2 still continued to segregate. One of these spore isolates was analyzed, and segregation was found to continue for more than 150 generations after spore germination. Whereas up to more than 80% of zygote clones in certain crosses were uniform, only 2 out of 91 tetrads were uniform, i.e. all four spores were homoplasmic for the same mitochondrial genotype. Presence or absence of recombinant mitochondrial phenytypes among secondary spore clones from tetrads indicated, whether, cytoplasmic mixing had occurred in the original zygote or not. Within an ascus, the number of spores containing recombinant genotype(s) is a direct measure for the extent of cytoplasmic mixing in the zygote. In 82 tetrads analyzed, the number of tetrads with 0, 1, 2, 3, and 4 spores containing recombinant genotype(s) were 25, 37, 14, 5, and 1, respectively. In conclusion, the extent of cytoplasmic mixing at the cell stage before forespore membrane formation is highly variable.     

Confirmation of intraspecific crossing and single and joint segregation of biochemical loci ofVolvariella volvacea

Nineteen lines ofV. volvacea were obtained from worldwide sources and analyzed for allozyme activity. Twelve loci (Aat, Acp-2, Dia, Est-4, Gipi, Lap-1, Pep-GL, Pep-LLL-1, Pep-LLL-2, Pgm, Pgd, andSod) were monomorphic; 5 loci (Ada, Gpt, Mpi, Np, andPep-PAP) were polymorphic. Nine unique genotypes were distinguished among the 19 lines. Multilocus enzyme electrophoresis was used to confirm intraspecific crosses between putative homokaryons. Putative homokaryons were recognized by electrophoresis of single-spore-derived cultures and used as breeding stock. These breeding stocks were grown together in dual culture on agar, and selections were made from the interaction zone. These selections were then transferred to make spawn. Intraspecific crosses were confirmed by the presence of heteromeric allozymes. To determine single and joint segregation of biochemical loci, 84 single-spore-derived offspring from a confirmed hybrid sporocarp with five heterozygous loci were analyzed. Mendelian segregation was verified for the five loci. One linkage (Np withGpt,r=0.0) was found among 10 pairwise comparisons for joint segregation. The basidiospores ofV. volvacea are haploid and homokaryotic isolates may mate to yield heterokaryons.     

Glucosamine resistance in yeast

Summary Two cytoplasmic, glucosamine resistant (GR) mutants of Saccharomyces cerevisiae, GR6 and GR10, were crossed to strains bearing known mitochondrial markers. Analysis of vegetative and meiotic segregation patterns in these crosses suggested that the glucosamine resistance conferring factor did not reside on mitDNA. This was confirmed by ethidium bromide treatments which completely abolished oligomycin resistance due to a mitochondrial mutation at the OLI2 locus but which failed to eliminate the GR factor present in the same strain. Comparison of GR6 and GR10 to some other known cytoplasmic determinants suggested that while glucosamine resistance is not related to the killer plasmid it may be allelic to the URE3 determinant and/or to the PSI factor.     

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.