Sensitive Progeny Research Articles

Mapping QTLs for Reproductive Stage Salinity Tolerance in Rice Using a Cross between Hasawi and BRRI dhan28.

Salinity stress is a major constraint to rice production in many coastal regions due to saline groundwater and river sources, especially during the dry season in coastal areas when seawater intrudes further inland due to reduced river flows. Since salinity tolerance is a complex trait, breeding efforts can be assisted by mapping quantitative trait loci (QTLs) for complementary salt tolerance mechanisms, which can then be combined to provide higher levels of tolerance. While an abundance of seedling stage salinity tolerance QTLs have been mapped, few studies have investigated reproductive stage tolerance in rice due to the difficulty of achieving reliable stage-specific phenotyping techniques. In the current study, a BC1F2 mapping population consisting of 435 individuals derived from a cross between a salt-tolerant Saudi Arabian variety, Hasawi, and a salt-sensitive Bangladeshi variety, BRRI dhan28, was evaluated for yield components after exposure to EC 10 dS/m salinity stress during the reproductive stage. After selecting tolerant and sensitive progeny, 190 individuals were genotyped by skim sequencing, resulting in 6209 high quality single nucleotide polymorphic (SNP) markers. Subsequently, a total of 40 QTLs were identified, of which 24 were for key traits, including productive tillers, number and percent filled spikelets, and grain yield under stress. Importantly, three yield-related QTLs, one each for productive tillers (qPT3.1), number of filled spikelets (qNFS3.1) and grain yield (qGY3.1) under salinity stress, were mapped at the same position (6.7 Mb or 26.1 cM) on chromosome 3, which had not previously been associated with grain yield under salinity stress. These QTLs can be investigated further to dissect the molecular mechanisms underlying reproductive stage salinity tolerance in rice.

Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus.

A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique ‘bulk segregant analysis’ (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.

Elimination of common scab sensitive progeny from a potato breeding population using thaxtomin A as a selective agent

Common scab is one of the most important soil‐borne diseases of potato and is difficult to control. Selection of potato breeding lines for resistance to common scab is also cumbersome due to environmental factors influencing symptom development and an erratic spatial distribution of the scab pathogens (Streptomyces spp.) in the field. The bacterial phytotoxin thaxtomin A, which causes scab symptoms, can be used to screen large numbers of potato seedlings for tolerance in vitro, but few studies have investigated whether the results correspond to resistance to common scab observed in the field. In this study, 120 F1 potato progeny from a single cross were screened in vitro by exposing the seedlings to thaxtomin A added to the culture medium. Eighteen genotypes were selected based on high sensitivity or tolerance using shoot growth as the criterion, multiplied in vitro, and tested for resistance to common scab caused by S. turgidiscabies and S. scabies in a glasshouse and in three different fields. Evaluation of ca. 6500 tubers showed that the 18 potato genotypes differed in scab indices and disease severity (P < 0·0001). The relative shoot height in vitro (thaxtomin A used at 0·5 μg mL−1) and the scab index in the field showed significant correlation (rs = −0·463, P = 0·0528, n = 18), also consistent with the results obtained under controlled conditions in the glasshouse. Hence, the in vitro bioassay may be used to discard scab‐susceptible genotypes and elevate the overall levels of common scab resistance in the potato breeding populations.

Inheritance of Cross-sensitivity in Sweet Corn to Herbicides Applied Postemergence

Some sweet corn (Zea mays L.) hybrids and inbreds can be severely injured by applications of postemergence herbicides. An association was observed between the responses of sweet corn hybrids and inbreds to nicosulfuron and mesotrione, and F2 families derived from a cross of a sensitive (Cr1) and a tolerant (Cr2) sweet corn inbred segregated for response to these two herbicides. These observations prompted us to examine the inheritance of sensitivity in sweet corn to multiple postemergence herbicide treatments with different modes of action and to determine if there was a common genetic basis for cross-sensitivity to these herbicides. The sensitive and tolerant inbreds, progeny in the F1, F2, BC1, and BC2 generations, and BC1S1, BC2S1, F2:3 (S1:2) and F3:4 (S2:3) families were screened for responses to eight herbicide treatments. Based on segregation of tolerant and sensitive progeny and segregation of family responses, our data indicate that a single recessive gene in Cr1 conditioned sensitivity to four acetolactate synthase (ALS)-inhibiting herbicides (foramsulfuron, nicosulfuron, primisulfuron, and rimsulfuron), a 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide (mesotrione), a growth regulator herbicide combination (dicamba + diflufenzopyr), and a protoporphyrinogen oxidase (PPO)-inhibiting herbicide (carfentrazone). Based on highly significant positive correlations of phenotypic responses among BC1S1, BC2S1, F2:3, and F3:4 families, the same gene (or closely linked genes) appeared to condition responses to each of these herbicide treatments. The dominant allele also conditions tolerance to bentazon [a photosystem II (PSII)-inhibiting herbicide] although another gene(s) also appeared to affect bentazon tolerance.

Genetic Analysis of Sensitivity to a Pyrenophora tritici-repentis Necrosis-Inducing Toxin in Durum and Common Wheat

ABSTRACT The fungus Pyrenophora tritici-repentis produces a toxin (Ptr ToxA) that causes rapid cell necrosis in sensitive wheat genotypes. A single recessive gene (tsn1) on chromosome 5BL in common wheat confers insensitivity to this toxin. Our objectives were to analyze the allelic relationships of genotypes that have shown insensitivity to a P. tritici-repentis necrosis-inducing toxin, map the gene for insensitivity to the necrosis-inducing factor produced by P. tritici-repentis in a durum wheat population, and determine the reaction to P. tritici-repentis of aneuploid genotypes that do not contain the gene. Greenhouse-grown plants of seven populations from crosses of insensitive genotypes; an F(2) population of durum wheat; and 'Chinese Spring' aneuploid, substitution, and deletion lines were infiltrated with Ptr ToxA. All crosses involving insensitive genotypes failed to produce sensitive progeny, indicating that the same gene is present in these genotypes. The gene for insensitivity in the durum population was mapped to the same region on 5BL as in common wheat using restriction fragment length polymorphism markers. 'Chinese Spring', its homoeologous group 5 nullisomic-tetrasomic stocks, and 5BL deletion lines were insensitive to the toxin. Substitution of a 5B chromosome from sensitive genotypes into 'Chinese Spring' resulted in sensitivity. Therefore, insensitivity is not conferred by a gene product per se, but rather conferred by absence of a gene for sensitivity.

Genetic control of the resistance of Erysiphe graminis f.sp. hordei to five triazole fungicides

The genetics of resistance to ergosterol demethylation inhibitor (DMI) fungicides of the triazole (conazole) group was examined in a cross between two isolates of the barley powdery mildew fungus, Erysiphe graminis (= Blumeria graminis) f.sp. hordei. One isolate, E1, was previously identified as being resistant to the triazole fungicide triadimenol, while the other, HL3/5, was sensitive. The 56 progeny tested were classified into two distinct groups, either being resistant to triadimenol, like E1, or sensitive, like HL3/5. The segregation ratio was not significantly different from 1:1, consistent with responses to triadimenol being controlled by a single gene. In further tests with cyproconazole, epoxiconazole, propiconazole and tebuconazole, all the progeny classified as resistant to triadimenol were also more resistant to each of these other triazole fungicides than were any of the triadimenol‐sensitive progeny. This is consistent with the triadimenol resistance allele also conferring cross‐resistance to the other triazoles. The ratio between the responses of the resistant and sensitive progeny (the resistance factor, RF) was greatest for triadimenol, followed by tebuconazole, propiconazole, epoxiconazole and cyproconazole, in that order. The RF for triadimenol was much greater when the fungicide was applied as a seed treatment than when it was sprayed. Five isolates, covering the five levels of responses to triadimenol identified previously in the UK population of E. graminis f.sp. hordei, were used as standards; a triadimenol‐sensitive isolate and one with the lowest level of resistance were sensitive to all four of the other fungicides, while three isolates with higher levels of triadimenol‐resistance were also more resistant to the other chemicals.

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line.

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0-93 ⋅ 10-6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10-6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10-6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1.

Mating Behavior in Field Populations of Monilinia fructicola

The mating behavior of Monilinia fructi? cola was evaluated by examining the single ascospore progeny from 82 apothecia collected in the San Joa- quin Valley of California. The results show that indi? vidual apothecia generated populations of genetically diverse progeny as defined by the segregation of my? celial incompatibility genes. Furthermore, a number of apothecia segregated a 1:1 ratio of benomyl resis? tant and sensitive progeny. These results indicate that M. fructicola is capable of outcrossing and generating new genotypic combinations during each spring as apothecia are produced.

Mating behavior in field populations of Monilinia fructicola

The mating behavior of Monilinia fructi? cola was evaluated by examining the single ascospore progeny from 82 apothecia collected in the San Joa- quin Valley of California. The results show that indi? vidual apothecia generated populations of genetically diverse progeny as defined by the segregation of my? celial incompatibility genes. Furthermore, a number of apothecia segregated a 1:1 ratio of benomyl resis? tant and sensitive progeny. These results indicate that M. fructicola is capable of outcrossing and generating new genotypic combinations during each spring as apothecia are produced.

Development of Two Screening Methods for Tomato Seedlings Containing the NPT II Selectable Marker Gene

Two simple, cost-effective methods to screen fresh-market tomato seedlings containing the kanamycin resistance gene construct, in which the nopaline synthase promoter from pMON128 is driving the NPT II gene, have been developed. The assays can reliably distinguish kanamycin-resistant from sensitive progeny for a variety of tomato genotypes. One method is an in vitro germination assay. Two selective agents, geneticin (G418) and kanamycin sulfate, were evaluated for their efficacy, and titrations were performed to determine the optimal concentration of the appropriate agent. The second method is a whole-plant spray test of seedlings to identify kanamycin-resistant progeny. A protocol was developed that could distinguish positives from negatives in 5 weeks. Currently, these assays are being used to screen R1 progeny rapidly to identify positives and obtain segregation ratios. They also are being used to screen R2 progeny to identify quickly those lines that are “true-breeding” or homozygous for field trial evaluation.

Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis.

In several plant systems expression of structurally intact genes may be silenced epigenetically when a transgenic construct increases the copy number of DNA sequences. Here we report epigenetic silencing in Arabidopsis lines containing transgenic inserts of defined genetic structure, all at the same genomic locus. These comprise an allelic series that includes a single copy of the primary insert, which carries repeated drug resistance transgenes, and a set of its derivatives, which as a result of recombination within the insert carry different numbers and alleles of resistance genes. Although the drug resistance genes remained intact, both the primary and some recombinant lines nevertheless segregated many progeny that were partly or fully drug-sensitive because of silencing. As in other systems silencing was reversible, and correlated with decreased steady-state mRNA and increased DNA methylation. Each different number and combination of genes, on the same or different (i.e., homologous) chromosomes, conditioned its own idiosyncratic segregation pattern. Strikingly, lines with a single gene segregated only a few slightly drug-sensitive progeny whereas multi-gene lines segregated many highly sensitive progeny, indicating dependence of silencing at this locus on repeated sequences. This argues strongly against explanations based on antisense RNA, but is consistent with explanations based on ectopic DNA pairing. One possibility is that silencing reflects the interaction of paired homologous DNA with flanking heterologous DNA, which induces condensation of chromatin into a non-transcribable state.

Sensitivity to an Ethylene Biosynthesis-Inducing Endoxylanase in Nicotiana tabacum L. cv Xanthi Is Controlled by a Single Dominant Gene.

The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). Sensitivity to EIX is controlled by a single dominant gene, based on chi-square analysis of segregation ratios.

Stable transformation of the moss Physcomitrella patens

We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Fate of DNA encoding hygromycin resistance after meiosis in transformed strains of Gibberella fujikuroi (Fusarium moniliforme)

Stability of foreign DNA transformed into a novel host is an important parameter in decisions to permit the release of genetically engineered microorganisms into the environment. Meiotic instability of transformed DNA has been reported in fungi such as Ascobolus, Aspergillus, and Neurospora. We used strains of Gibberella fujikuroi (Fusarium moniliforme) transformed with the hygr gene from Escherichia coli to study meiotic stability of foreign DNA in this plant pathogenic fungus. Crosses with single-copy transformants segregated hygr:hygs in a 1:1 manner consistent with that expected for a Mendelian locus in a haploid organism. Multicopy transformants, however, segregated hygr:hygs in a 1:2 manner that was not consistent with Mendelian expectations for a chromosomal marker, even though two unrelated auxotrophic nuclear genes were segregating normally. Segregation ratios in crosses in which hygr was introduced via the male parent did not differ significantly from crosses in which the transformed strain served as the female parent. Some of the sensitive progeny from the crosses with the multicopy transformants carried hygr sequences. When these phenotypically sensitive progeny were crossed with a wild-type strain that carried no hygr sequences, some of the progeny were phenotypically hygr. Some progeny from some crosses were more resistant to hygromycin than were their sibs or the transformant strains that served as their parents. Transformants passaged through a maize plant only rarely segregated progeny with the high levels of resistance. The mechanism underlying these genetic instabilities is not clear but may involve unequal crossing over or methylation or both. Further work with cloned genes with homology to sequences already present in the Fusarium genome is warranted.

Two changes of the same nucleotide confer resistance to diuron and antimycin in the mitochondrial cytochrome b gene of Schizosaccharomyces pombe

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and antimycin, both inhibitors of mitochondrial respiration, block electron flow between cytochromes b and c 1. Mutants resistant to either drug have been selected using Schizosaccharomyces pombe strains with an extrachromosomally inherited mutator. In analogy to Saccharomyces cerevisiae these mutational sites were assumed to map in the cytochrome b gene. DNA sequence analysis showed that two changes in the same nucleotide are responsible for resistance to antimycin and diuron. Analysis of resistant and sensitive progeny of crosses between the mutants and the wild type confirmed the correlation between mutational alteration and resistant phenotype.

Studies on the inheritance of resistance to metalaxyl in Bremia lactucae and on the stability and fitness of field isolates

One hundred and eighty‐one sexual progeny isolates from three F1, one F2, five BC1 and one BC2 generations of crosses involving two metalaxyl‐resistant and two metalaxyl‐sensitive isolates of Bremia lactucae were assessed for response to metalaxyl. In most cases, isolates were also tested for virulence phenotype and sexual compatibility type. Isolates could be classified into three phenotypes (sensitive, resistant and intermediate) on the basis of their response to fungicide. Sensitive progeny isolates were similar to the sensitive parents and did not sporulate on lettuce seedlings grown in the presence of > 1.0 μg/ml metalaxyl. Resistant progeny isolates, in common with the resistant parents, sporulated as readily on lettuce seedlings grown in the presence of 100 μg/ml metalaxyl as on untreated seedlings. Progeny isolates which expressed an intermediate phenotype not observed in field samples were also recovered. This phenotype was more variable but isolates so classified were able to sporulate on seedlings grown in the presence of 100 μg/ml metalaxyl but exhibited a latent period (time from inoculation to appearance of sporulation) which was on average twice as long as that observed on seedlings grown in the absence of fungicide.Segregation patterns suggested that response to metalaxyl is controlled at a single locus exhibiting incomplete dominance but that the expression of response in the heterozygous condition may be affected by modifier genes. There was evidence that the metalaxyl‐resistant parental field isolates were heterokaryotic (possibly carrying nuclei homozygous and heterozygous at the locus controlling metalaxyl response). After 10‐30 asexual generations in the absence of fungicide, the response to metalaxyl of resistant field isolates began to approach that of an intermediate type. This phenomenon did not occur with a resistant sexual progeny isolate (presumed homokaryotic) and is additional evidence for the heterokaryosis of field isolates. The relative fitness of a range of isolates was examined by observing changes in the proportions of spores over three asexual generations after they were initially mixed in a 1:1 ratio with mutant isolates carrying a microscopically visible genetic marker (large lipid droplets). Most field isolates were more fit than the mutant isolates used, regardless of whether they were resistant or sensitive to metalaxyl, and there was no evidence that resistant isolates carried any fitness deficit in comparison with the sensitive isolates examined. In contrast, sexual progeny isolates (particularly from BC1 generations) were often less fit than the mutant isolates used, but there was no evidence that this was associated with response to fungicide. These findings are discussed in relation to the mode of action of metalaxyl and disease control strategy.

Transformed callus does not necessarily regenerate transformed shoots

Agrobacterium tumefaciens carrying a disarmed Ti plasmid vector was used for the transformation of flax tissue. Transformed callus was obtained from inoculated hypocotyl segments and healthy green shoots were regenerated from this callus. Nopaline assays on shoot tissue were positive for nopaline content if carried out soon after removal of the shoot from the callus but negative if carried out 2-3 weeks after removal. All of these shoots gave rise to kanaymcin sensitive progeny and were most likely escapes arising from non-transformed cells protected from the selective agent by transformed cells in the callus. Careful analysis of regenerated shoots from transformed callus is necessary in order to distinguish escapes from true transgenics.

Do transcriptional enhancers also augment DNA replication?

Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (SV40) core enhancer was ligated either adjacent to or 2.6 kb distant from the SV40 minimal origin of replication (ori) in both orientations. Plasmids were transfected into T antigen producing COS cells, and episomal DNA was harvested for quantitation of replication. Replication could be assessed either as accumulation of fmol of MboI sensitive progeny DNA, or as a transition in % DNA in replicated (MboI sensitive) versus unreplicated (DpnI sensitive) form. The two measures were related exponentially (r = 0.86). The SV40 enhancer augmented replication 1.5-10 fold. The effect was time dependent, distance dependent (only the adjacent enhancer locus stimulated replication), partially orientation dependent, and enhancer copy number independent. Phorbol ester did not affect replication. The heterologous glucocorticoid enhancer had no effect on replication. We conclude that the SV40 enhancer's cis-effect on replication seems to be dependent on the close proximity to the replication origin of specific homologous sequences within the enhancer, rather than a typical enhancer-like effect.

Tolerance trials with young cocoa trees infected with cocoa swollen‐shoot virus

SUMMARYIn three short‐term field trials, 24 cocoa progenies were assessed for sensitivity to infection with a severe Ghanaian strain of cocoa swollen‐shoot virus. Some of the progenies were derived from tolerant parents, others had themselves shown mild symptoms as young seedlings. Stem diameters, diameter increments, canopy scores and latent periods (time between inoculation and expression of symptoms) of plants infected when 12 to 22 months old were compared with similar measurements of healthy plants.During the period between 6 and 18 months after inoculation, infection reduced the stem diameter increment of the least sensitive progeny in each trial by 52, 25 and 7%, the last value increasing to 38% during the period between 24 and 30 months after inoculation. Within trials, the effects of virus infection on stem diameter increments and canopy scores were well correlated but latent periods were not significantly correlated with any other variate.In one trial, pod yield was more closely related to canopy condition at the time of cropping than to stem diameter increment 2 yr earlier. Weak correlations between pre‐ and post‐bearing increases in diameters in these trials indicated that tolerance was not directly related to vigour. None of the progenies is considered suitable for direct use as tolerant varieties in Ghana.

Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardtii.

Analysis of the streptomycin resistant mutants recovered from control and N-methyl-N′-nitro-N-nitrosoguanadine (MNNG) treated haploid cultures of C. reinhardtii reveal that approximately 60% of the mutants are of the sr-1 type known to show Mendelian inheritance while 40% are of the sr-2 and sm-3 types known to be inherited in a uniparental (UP) manner. In contrast, most if not all streptomycin mutants recovered from similarly treated diploid cultures of C. reinhardtii are of the UP variety. Failure to recover sr-1 mutants from the diploid stock is explained by our findings that diploids heterozygous for Mendelian streptomycin resistance (sr-1/sr-1 +) are both stable and sensitive to streptomycin. Efficient recovery of UP streptomycin resistant mutants from diploids can be explained by the observations of Gillham (1963a, 1969) which demonstrate that diploids heterozygous for an sr-2 mutation (sr-2/sr-2 +) segregate resistant and sensitive progeny during mitotic cell division. The utility of diploids for isolating new UP mutant genotypes, for establishing the cellular localization of the UP genome(s), and for characterizing the rules governing UP gene segregation is discussed.