Monogenic Mutant Research Articles

In silico screening of a saturated mutation library of tomato.

A comprehensive mutant population is a basic resource for exploring gene function. We developed an isogenic tomato 'mutation library' in the genetic background of the inbred variety M82. A total of 13 000 M(2) families, derived from EMS (ethyl methane sulfonate) and fast-neutron mutagenesis, were visually phenotyped in the field and categorized into a morphological catalog that includes 15 primary and 48 secondary categories. Currently, 3417 mutations have been cataloged; among them are most of the previously described phenotypes from the monogenic mutant collection of The Tomato Genetics Resource Center, and over a thousand new mutants, with multiple alleles per locus. The phenotypic database indicates that most mutations fall into more than a single category (pleiotropic), with some organs such as leaves more prone to alterations than others. All data and images can be searched and accessed in the Solanaceae Genome Network (SGN) on a site called 'The Genes That Make Tomatoes' (http://zamir.sgn.cornell.edu/mutants/).

Inheritance of plant height and allelism tests of the dwarfing genes in Chinese barley

AbstractIn order to find new dwarfing genes, the inheritance of plant height in 25 Chinese barley dwarf accessions was studied and allelism tests carried out, not only between the dwarfing genes found but also with the known dwarfing genes uz, br, sdw and denso. The results showed that out of the 25 dwarf accessions, 20 were due to monogenic recessiveness and four to digenic recessiveness. Only the short plant character in ‘1974E’ was controlled by a recessive together with a dominant dwarfing gene. In the present study, seven recessive and one dominant new dwarfing genes were identified. Five recessive genes were found in the monogenic mutants ‘91G318’, ‘91D27’ and ‘93‐597’ and in the Tibetan monogenic dwarf landraces ‘Jia Jiu’ and ‘BQK’, respectively. The other two recessives were found in the Tibetan digenic dwarf landraces ‘ZLL’ and ‘ZLLQK’, and the one dominant gene in the digenic mutant ‘1974E’.

Changes in photoperiod or temperature alter the functional relationships between phytochromes and reveal roles for phyD and phyE.

The phytochromes are one of the means via which plants obtain information about their immediate environment and the changing seasons. Phytochromes have important roles in developmental events such as the switch to flowering, the timing of which can be crucial for the reproductive success of the plant. Analysis of phyB mutants has revealed that phyB plays a major role in this process. We have recently shown, however, that the flowering phenotype of the phyB monogenic mutant is temperature dependent. A modest reduction in temperature to 16 degrees C was sufficient to abolish the phyB mutant early-flowering phenotype present at 22 degrees C. Using mutants null for one or more phytochrome species, we have now shown that phyA, phyD, and phyE, play greater roles with respect to phyB in the control of flowering under cooler conditions. This change in the relative contributions of individual phytochromes appears to be important for maintaining control of flowering in response to modest alterations in ambient temperature. We demonstrate that changes in ambient temperature or photoperiod can alter the hierarchy and/or the functional relationships between phytochrome species. These experiments reveal new roles for phyD and phyE and provide valuable insights into how the phytochromes help to maintain development in the natural environment.

Physiological characterization of exaggerated-photoresponse mutants of tomato

Summary Four monogenic mutants in tomato ( Lycopersicon esculentum Mill.), i.e. three recessive mutations, high-pigment-1 ( hp-1 ), high-pigment-2 ( hp-2 ), and atroviolacea ( atv ), and one dominant mutation, Intense pigmentation ( Ip ), were used in this study. These mutants all show exaggerated photoresponses during deetiolation and seedlings having shorter hypocotyls and higher anthocyanin levels. The hp-1 and hp-2 have higher chlorophyll levels in immature fruit, giving them a dark green colour. Spectrophotometrical and immunological analyses of phytochrome A and phytochrome B revealed no differences between the mutants and the wild types (WTs), suggesting that the mutants are not photoreceptor mutants. Both hp-1 and hp-2 accumulate high levels of anthocyanin in continuous blue (B) and red (R) broad-band light. In contrast, atv has a WT level of anthocyanin in B and an exaggerated response in R. The Ip mutant has the opposite response: a WT level of anthocyanin in R and an exaggerated response in B. In B and R pretreat-ment studies, all mutants show an enhanced R/far-red light (FR)-reversible response compared with WT, but the Ip mutant shows a preferentially enhanced response in B. The hp-1 mutant exhibits a strong amplification of both the low fluence rate response and high irradiance response components of anthocyanin biosynthesis in red light. The atv mutant shows the strongest amplification of the HIR component. The Ip mutant exhibits an exaggerated anthocyanin response in B. All four mutants exhibit a normal elongation response to supplementary FR during the daily photoperiod.

Isolation and genetic analysis of ethanol-sensitive mutants of thermotolerant Kluyveromyces marxianus

Six monogenic mutants of K. marxianus, affected in their ability to tolerate ethanol, were assigned to 6 loci etr1 through etr6, probably the hot spots for ethyl methane sulfonate as these loci were found mutated in each of the non-monogenic mutants as well. Differential ethanol tolerance of allelic hexagenic mutants suggests that even more than 6 genes may be involved in controlling ethanol tolerance in K. marxianus.

Molecular Markers and Germplasm Enhancement: A Case Study in Tomato

The C.M. Rick Tomato Genetic Resources Center (TGRC) is a genebank of wild relatives, monogenic mutants, and miscellaneous genetic stocks of tomato. The wild species group includes representatives of all nine Lycopersicon spp., as well as four related Solanum species. One of the roles of the TGRC has been to foster the use of the widest available gene pool for tomato researchers. The wild nightshade Solanum lycopersicoides possesses a number of potentially useful traits, but has been untapped by breeders because of sterility and incompatibility barriers. We are using molecular markers to identify alien chromosomal segments introgressed from S. lycopersicoides into tomato. This project involves development of RFLP, RAPD, and isozyme marker linkage maps and their use in selection of homozygous segmental substitutions in backcross inbred progenies. In this fashion, a large proportion of the S. lycopersicoides genome has been integrated into the cultivated tomato. This study has also provided information on the nature of sterility and novel variation in hybrid derivatives.

Genetic variation in flowering time inArabidopsis thaliana

An inventory of naturally occuring differences in flowering time among ecotypes and induced monogenic mutations is presented and discussed. In general, there are two groups of genes among the induced monogenic mutants; responsive or less responsive to environmental factors. The interaction between flowering time genes is being studied by analysing the double mutants. Flowering is not abolished or even extremely delayed in any of the constructed double mutants: apparently these genes modify the process but are not essential. Many genes influence the transition to flowering in a quantitative way. All late/early flowering mutants isolated to date might have an effect on a floral repressor, possibly encoded for by theEMFpathway, which is either promotive or repressive.

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta, were analysed by two‐dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20‐day‐old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa, and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta. Crosses were made between this line and the wild type Landsberg erecta. F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.

The effect of daylength on the transition to flowering in phytochrome‐deficient, late‐flowering and double mutants of Arabidopsis thaliana

The effect of daylength on flowering was investigated in the following mutants of Arabidopsis thaliana: phytochrome B deficient (hy3=phyB); phytochrome chromophore deficient (hy2); late‐flowering (co, gi. fca and fwa); the hy2 and hy3, late‐flowering double mutants and the hy2, hy3, late‐flowering triple mutants. The hy mutants flower with fewer rosette leaves than the Landsberg erecta wild type under both long day and short day conditions and express this effect to a different degree in all late‐flowering mutant backgrounds and under both daylengths, with the exception of fca under short days. The number of cauline leaves and days to flowering is less affected by the hy genotype. The hy2, hy3 double mutants flower with even fewer rosette leaves than the hy2 and hy3 monogenic mutants, suggesting an inhibitory role for phytochrome B and other stable phytochromes on flowering. The complex interaction between phytochrome, daylength and the effect of the late‐flowering genes on the various parameters that describe the transition to flowering in Arabidopsis is discussed.

A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White-hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups. The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild-type cell walls contained about equal amounts of mannose and glucose. This indicates that both low-mannose and low-glucose cell wall mutants had been obtained. Further characterization showed the presence of three low-mannose cell wall mutants with a mnn9-like phenotype, affected, however, in different genes. In addition, four new killer-resistant (kre) mutants were found, which are presumably affected in the synthesis of beta 1,6-glucan. Most low-glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of beta 1,3-glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with beta 1,3-glucan synthesis, and four cwh mutants were temperature-sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.

An Arabidopsis thaliana RFLP mapping set to localize mutations to chromosomal regions

SummaryMapping of newly identified Arabidopsis thaliana mutants is an important step towards their molecular characterization and the attempt to saturate the genome by known mutations. The classical genetic analysis using phenotypic tester lines is well‐established, but laborious, time‐consuming and potentially ambiguous. An alternative molecular strategy was developed that is based on RFLPs. Subcloned DNA markers that detect only segregating RFLP bands distinguishing A. thaliana ecotype Landsberg from Columbia or Enkheim after EcoRI restriction digestion compose an Arabidopsis RFLP mapping set (ARMS). Up to 13 markers uniformly cover the five A. thaliana chromosomes and can be scored in only two successive Southern experiments on a single blot without mutual interference of the signals. Thus, this system allows a simple, reliable, rapid and especially inexpensive mapping of any monogenic mutant locus to the A. thaliana chromosomes. Several loci can be analysed in one experiment if the respective blots are hybridized together. This paper demonstrates the mapping of two recessive mutants affecting the development of A. thaliana leaves which had been generated in the Columbia and Enkheim ecotype by analysing less than 20 F2 individuals. Further markers to refine or verify the result on the same blot can be chosen out of 14 additional probes detecting single segregating EcoRI polymorphic bands as well.

Abscisic acid-insensitive mutations provide evidence for stage-specific signal pathways regulating expression of an Arabidopsis late embryo genesis-abundant (lea) gene

An Arabidopsis homolog of the abscisic acid (ABA)-inducible cotton D19 and wheat Em genes was cloned and its expression assayed at two developmental stages in wild-type, ABA-deficient (aba) and three ABA-insensitive (abi) lines of Arabidopsis thaliana. Expression of this gene was reduced slightly in seeds of aba mutants and approximately ten-fold in abi3 mutants, but seed expression was not decreased in either abi1 or abi2 monogenic mutants. In contrast, the abi1 and abi2 mutants showed a very slight reduction of ABA inducibility in 8-day-old plants, while the responses of aba and abi3 mutants were comparable to that of wild type. Although previous studies have shown that none of the abi mutations show completely stage-specific effects, the results reported here indicate that the importance of each of the ABI loci in regulating this single gene is stage-dependent. Furthermore, the fact that none of the abi mutations show more than minor effects on exogenous ABA inducibility of the Arabidopsis D19/Em homolog in young plants suggests that an additional ABA signalling pathway may be operating during vegetative growth.

Genetic basis of plant regeneration in hexaploid triticale

Glume cultures of monogenic recessive mutants PGTSLS1 (Plant Genetics triticale selection large spike 1) and PGTSLG2 (Plant Genetics triticale selection long grain 2) were employed along with their parent PGTS control (Plant Genetics triticale selection control) and their F1 and F2 progenies, to determine the genetic basis of plant regeneration in hexaploid triticale. The mutant PGTSLS1 exhibited greater efficiency of plant regeneration (22.4%) followed by PGTS control (7.6%) and PGTSLG2 did not exhibit any regeneration. All the three F1's exhibited plant regeneration frequency on par with that of control (6.9–7.3%), suggesting dominant nature of control genotype over the isogenic mutants. The F2 results suggested that genetic control over the high frequency regeneration of PGTSLS1 was monogenic recessive in nature, and genetic control over the recalcitrant nature of PGTSLG2 also was monogenic recessive. The F2 of the cross PGTSLS1 × PGTSLG2 segregated into four classes. Of the 114 F2 plants, 19 showed no regeneration, 70 of them exhibited 6–8% regeneration, 20 of them 19–24% regeneration, and 5 of them exhibited highest frequency regeneration (57–60%). These observations suggest dihybrid segregation for regeneration. The highest frequency of plant regeneration (57–60%) exhibited by 5 F2 plants may be due to the interaction of non-allelic genes in recessive condition. These results clearly demonstrate the association of at least two genes with plant regeneration in hexaploid triticale.

A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana

Monogenic mutants of the early ecotype Landsberg erecta were selected on the basis of late flowering under long day (LD) conditions after treatment with ethyl methanesulphonate or irradiation. In addition to later flowering the number of rosette and cauline leaves is proportionally higher in all mutants, although the correlation coefficient between the two parameters is not the same for all genotypes. Forty-two independently induced mutants were found to represent mutations at 11 loci. The mutations were either recessive, intermediate (co locus) or almost completely dominant (fwa locus). The loci are located at distinct positions on four of the five Arabidopsis chromosomes. Recombinants carrying mutations at different loci flower later than or as late as the later parental mutant. This distinction led to the assignment of eight of the loci to three epistatic groups. In wild type, vernalization promotes flowering to a small extent. For mutants at the loci fca, fve, fy and fpa, vernalization has a large effect both under LD and short day (SD) conditions, whereas co, gi, fd and fwa mutants are almost completely insensitive to this treatment. SD induces later flowering except for mutants at the co and gi loci, which flower with the same number of leaves under LD and SD conditions. This differential response of the mutants to environmental factors and their subdivision into epistatic groups is discussed in relation to a causal model for floral initiation in Arabidopsis thaliana.

La morphogenèse florale chez le pétunia. III. Déviations dans le programme organogène chez un mutant femelle stérile

A monogenic mutant of Petunia hybrida R-n57 exhibits flowers with abnormal gynoecium: various malformations, such as the opening of the style and the nonprotection of ovules, or the masculinization with development of pollen sacs, anthers, and male gametophyte growing with or within ovules. The same type of abnormalities happens over and over along one inflorescential axis so that an ontogenic analysis can be conducted. Some assumptions are supported by the histocytological study: a few punctual modifications of the normal organogenesis program, recently established, and one new stage ending at the masculine or abnormal gynoecium, with homeotic members, are involved. Relations between the deflected development and female sterility are argued. Key words: flower, teratology, morphogenesis, mutant, Petunia.

Use of Biotechnology to Create or Transfer Novel Traits in Tomato

The tomato (Lycopersicon esculentum) is often cited as an example of success in plant breeding and of potential for further improvement through use of biotechnology. The interest in tomato as a model system for genetic engineering is due in part to the large body of work done on the genus Lycopersicon over the past 50 years. This work includes the collection of germplasm of L. esculentum and its wild relatives, creation of chromosome addition and translocation stocks, the discovery or creation of >1200 monogenic mutants (Stevens and Rick. 1986). transfer from wild species of dis ease resistance genes. the creation of near-isogenic lines for a num ber of mutants or resistance genes, and the development of the clas sical genetic map (Tanksley et al., 1990). which has >300 markers including mutants, isozymes and resistance genes. The attractiveness of tomato as a research system also is due to the characteristics of the species that lend it to genetic work. L. esculentum and its wild relatives are diploid species, 2N=24, and are amenable to cyto logical study. L. esculentum is easy to self-pollinate or cross, resulting in relatively high seed set. L. esculentum has a relatively small genome (0.7 pg). which has remarkably few duplicate loci (Rick, 1971; Tanksley et al., 1987). An excellent central resource on the cultivation, genetics, and biology of L. esculentum and its wild relatives is the book “The Tomato Crop” (Astherton and Rudich, 1986). Lists of the plant materials available in Lycopersicon can be found in the yearly publication Reports of the Tomato Genetic Cooperative. In a recent article. Hille et al. (1989) summarized the use of biotechnology, in the broadest sense of the term, in tomato improvement. Rather than repeat material in that excellent article. the focus of this discussion is to overview the pos sibilities and the potential value of the use of emerging technologies for tomato improvement. Progress to date in using molecular developments for plant im provement centers on two molecular techniques. The creation/use of RFLP maps and the introduction of foreign DNA into the plant genome using Ti-mediated gene transfer. If one also considers tis sue culture under the rubric of “biotechnology”, the possibility of plant improvement by protoplast fusion and regeneration also may be considered. The current tomato RFLP map is probably one of the most com plete maps of a higher plant genome (Tanksley et al., 1990). Once created. the RFLP map has several uses for plant improvement. The map can be used to locate and identify molecular makers for genes of interest (Young and Tanksely. 1989). Once closely linked molecular markers have been identified. the markers can be used in indirect screening for the gene of interest. and so facilitate the rapid transfer of desired major genes while minimizing linkage drag (Tanksley et al.. 1989; Tanksley, 1989). RFLP mapping can be used further to identify the regions of the genome that are associat ed with important quantitative traits. Once these regions are identi fied, the information can be used to facilitate the transfer of genes affecting the quantitative trait (Paterson et al., 1988; Tanksley et al.. 1989). The value of the RFLP map for single genes or quantitative traits is evident. These techniques are currently being used in a number of laboratories focusing on diverse traits. The disadvantages of the systems are the cost and time involved, which can be minimized by careful planning, and the need in tomato to use plants derived from interspecific crosses to acquire sufficient polymorphisms. However. the advantages of the systems definitely exceed the disadvantages.

Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses

Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways.

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.

Kinetics of L-valine uptake in tobacco leaf discs. Comparison of wild-type, the digenic mutant Valr-2, and its monogenic derivatives

Uptake rates of L-valine in epidermis-free leaf discs of tobacco (Nicotiana tabacum L. cv. Xanthi) were measured over the concentration range 0.1 μM to 50 mM. Wild-type tobacco was compared with the digenic mutant Val(r)-2 (genotype vr2/vr2; vr3/vr3), and with the monogenic mutant strains h9 and h10 (genotype +/+; vr3/vr3) and h17 and h23 (genotype vr2/vr2; +/+). Rate equations consisting of one to three Michaelis-Menten terms, possibly in combination with a linear term were fitted to the kinetic data. These rate equations are equivalent to rational polynomials which may be regarded as the general type of mathematical function describing the kinetics of enzymes and carriers. Kinetic data of the four genotypes conformed to the sum of three Michaelis-Menten terms. Accordingly, three kinetic components could be distinguished. In the wild-type the approximate Kms were 40 μM, 1mM, and 40 mM, respectively. In Val(r)-2 a component with a very low Km (about 4 μM) was found which may represent either the modified low-Km component of the wild-type or a fourth component which is undetectable in the wild-type by kinetic analysis. The Vmax of the low-Km component in Val(r)-2 was at least a 100-fold lower than in the wild-type. In the presence of one of the mutant genes the calculated Vmax of the low-Km component was 48% (strains h9 and h10) or 40% (strains h17 and h23) of the corresponding Vmax in the wild-type. It is reasoned that the mutations have no effect on the activity of the other two kinetic components, though the evidence for this is circumstantial. Autoradiographs of leaf discs showed that in Val(r)-2 the uptake of (14)C-labelled valine in both mesophyll and minor veins was strongly reduced as compared with the wild-type.

Ethanol-sensitive mutants of Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism. Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucose-supplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.