Euploid Hybrids Research Articles

Breaking a Species Barrier by Enabling Hybrid Recombination

Hybrid sterility maintains reproductive isolation between species by preventing them from exchanging genetic material. The genome sequences of the yeasts Saccharomyces paradoxus and Saccharomyces paradoxus have diverged by about 12%, yet they can mate and form viable diploid hybrids, which can reproduce asexually by mitosis. But these hybrids are sexually sterile: nearly all the gametes they produce by meiosis are inviable because they are aneuploid, lacking at least one essential chromosome. Chromosomes from the different species mis-segregate during hybrid meiosis due to anti-recombination; their sequences are too diverged to efficiently recombine and form crossovers. Previous methods to increase hybrid fertility by enhancing meiotic crossing over have had counteracting detrimental effects on asexual viability, due to increased mutagenesis and ectopic recombination. We were able to increase hybrid fertility 70-fold by suppressing two genes, MSH2 (YOL090W) and SGS1 (YMR190C), specifically during meiosis, whilst maintaining their mitotic expression. This confirms that anti-recombination is the principal cause of hybrid sterility. By overcoming this barrier, we were able to generate viable euploid hybrid gametes containing recombinant genomes from these two highly diverged parent species. We used this unique resource to map intrinsic Bateson-Dobzhansky-Muller incompatibilities contributing to hybrid gamete inviability. The ability to interbreed such diverged species will allow mapping of other intrinsic traits such as hybrid depression or vigour, extrinsic ecological adaptations that differentiate species, or commercially important characteristics.

Comparative meiosis and cytogenomic analysis in euploid and aneuploid hybrids of Urochloa P. Beauv.

The genus Urochloa includes most of the important grasses and hybrids currently used as pastures in the tropical regions. Cytogenetic analyzes have identified some aneuploid hybrids that provide new perspectives for genetic breeding. The objective was to analyze the meiotic behavior in euploid (2n = 4x = 36) and aneuploid (2n = 4x = 36 + 2) hybrids of U. ruziziensis x U. decumbens and U. ruziziensis x U. brizantha. Later, the chromosomes and respective genomes involved in pairing configurations and abnormalities were identified through GISH, with an emphasis on tracking the behavior of the additional chromosomes in the aneuploid hybrid U. ruziziensis x U. decumbens (B1B2B2B2 genomes). The aneuploid U. ruziziensis x U. decumbens shows a higher frequency of univalents, reduction of bivalents, and higher index of irregularities compared with the euploid hybrid. For the aneuploid U. ruziziensis x U. brizantha, there was a reduction in the frequency of univalents, an increase in bivalent and trivalent rates and a lower frequency of abnormalities when compared with the euploid hybrid. The rates of meiotic abnormalities and pairing configurations are parental genotype-dependent and influenced by trisomy. The chromosomes of the B1 and B2 genomes of the aneuploid hybrid (U. ruziziensis x U. decumbens) are involved in the formation of univalents, bivalents, and multivalents in inter-, intra- and inter-intragenomic pairings. In general, the segregation times of chromosomes of the genomes are different, since the chromosomes of the B1 genome segregate more slowly.

Ploidy Manipulations and Genetic Markers as Tools for Analysis of Quantitative Trait Variation in Progeny Derived from Triploid Plantains

Plantains (Musa spp. AAB group) are starchy bananas widely grown in Africa and tropical America by small landholders. Genetic analysis of the plantain genome is difficult due to its triploidy and high sterility. Ploidy manipulations (scaling up and down the number of chromosomes) and interspecific plantain-banana hybridization opened the path for the genetic amelioration of the crop and for the investigation of its genome for further manipulations. This report shows the associated effects of ploidy and of the major loci controlling vegetative fruit parthenocarpy and resistance to black sigatoka fungal leaf spot on growth and yield characteristics of plantain-banana euploid hybrids. The number of copies of the black sigatoka resistance allele (bs,) and of the fruit parthenocarpy gene (P,) in addition to their intralocus interaction and ploidy level were all found to significantly affect bunch and fruit characteristics in euploid plantain-banana hybrids. Epistasis significantly affected fruit weight and size in one cross but not in another. Significant multiple regression models combining ploidy and genetic markers explained 15 % to 85 YO of quantitative trait variation (QTV). The amount of QTV accounted by ploidy and genetic markers varied according to the characteristic and cross in which the markers were examined.

Chromosome Pairing Patterns in Interspecific Hovdeum Lechleri × H. Vulgare (Cultivated Barley) Hybrids with 2n = 21-29

Individual chromosomes or the whole genome of cultivated barley are often eliminated in interspecific Hordeum lechleri × H. vulgare crossing combinations (rarely one or a few chromosomes of H. lechleri are eliminated). This produces series of plants with chromosome numbers ranging from 2n = 21 (true trihaploids of H. lechleri and rarely aneuploids), through 2n = 22-27 (aneuploids) to 2n = 28, 29 and 30 (true hybrids and aneuploids). In such material the effect on meiotic pairing was studied for 1. influence of parental genotypes; 2. influence on pairing by chromosome number; and 3. influence on pairing by presence/absence of individual chromosomes identified by C-banding of root-tip cells. A total of 126 plants representing combinations of seven varieties of cultivated barley and ten accessions of H. lechleri were investigated. Overall there was a large variation in pairing between plants ranging from 0.12–10.09 chiasmata/cell. True trihaploids of H. lechleri (2n = 21) without barley chromosomes had a “basic pairing” ranging from 0.78–4.58 chiasmata/cell. In aneuploids and euploid hybrids the presence of individual barley chromosomes or the entire complement of barley generally resulted in higher pairing than in trihaploids. This contradicts previous proposals of a depressing effect of the H. vulgare genome in interspecific Hordeum hybrids. Some hybrid combinations showed a large difference in pairing according to parental origin. There seemed to be no clear tendencies of pairing related to different chromosomal numbers. The presence of chromosome 4H of barley had a negative effect on meiotic pairing. The greater part of the recorded pairing is assumed to reflect homoeologous pairing between the three genomes of H. lechleri whereas allosyndetic pairing between H. vulgare and H. lechleri chromosomes is considered low.

Cytological Study of Euploid and Monosomic Wheat-Rye Hybrids

The influence of male and female genotypes, the effects of their interactions, the influence of ID, 3D, 4D and 5B chromosomes and the effects of their interaction were studied in 30 F1 wheat - rye euploid and aneuploid hybrids / 2n =28, 27, A B D R / concerning 7 morphological characters: heading date, common tillering productive tillering, culm length, last internode length, ear length and spikelet number per ear. F1 hybrids were obtained by crosses between four hexaploid cultivars Triticum aestivum L.2n=42: Chinese Spring /CS/, Mironovka 808 /M 808 /, Zlatka /Z1 /, Novostepnyachka / N /, as well as their monosomic lines by 1D, 3D, 4D and 5B chromosomes with two inbred lines Secale cereale L. 2n = 14 [Bulgarian Low Stem Rye /BLSR/ and Lozen 14 / L 14 / The morphological characters of the parental genotypes were as well studied. It was established that the parental genotypes influence all of the studied characters, while the effect of their interaction was observed in 14.3 % of the characters (ear length). The group of the monosomic hybrids on 3D chromosomes shows an influence different from the other hybrids for 42.9% of the characters ( heading date, ear length, spikelet number per ear); the hybrids group on 5B chromosome - 71.4 % from the characters (heading date, common and productive tillering, last internode length, ear length); the group of the euploid hybrids - 85.7% of the characters (do not show a difference on culm length).

Influence of black Sigatoka disease on the growth and yield of diploid and tetraploid hybrid plantains

Plantain and banana ( Musa spp.) are important staple food crops in the humid forest and mid-altitude agro-ecologies of sub-Saharan Africa. Black Sigatoka, caused by Mycosphaerella fijiensis, is an airborne fungal disease which attacks the leaves of the Musa plant. It is commonly considered the major constraint to plantain production in West Africa. The disease causes severe leaf necrosis and reduces yield by 30 to 50%. Twenty euploid hybrids, derived from interspecific interploidy crosses were selected for a range of host responses to black Sigatoka. The performance of these hybrids was compared with that of their parents to determine the influence of the disease on growth parameters and components of yield. There were significant differences for all components of black Sigatoka resistance, growth parameters, and yield components among the hybrids. For diploid hybrids, which often had a short growth cycle, early flowering, or both, the disease incubation time was significantly correlated with days to fruit filling ( P<0.05). However, for tetraploid hybrids that had a long growth cycle and delayed flowering, the correlation was not significant ( P ≥ 0.05). For diploid and tetraploid hybrids, the disease evolution time and the disease development time were both correlated ( P < 0.05) with days to fruit filling. The bunch weight of tetraploid hybrids was correlated ( P < 0.05) with the disease development time that was easily scored by the youngest leaf spotted at flowering (ρ = 0.933; P < 0.001). Hence, black Sigatoka resistant hybrids with potential high yield could be selected efficiently by recording the youngest leaf spotted at flowering.

Genetic and phenotypic correlations in plantain-banana euploid hybrids

The objective of this work was to determine intraclass correlations for growth and yield traits, as well as associations between them, in segregating hybrids. Diploid, triploid and tetraploid hybrids were obtained from triploid-diploid crosses between plantains and bananas. These hybrids were evaluated as plants and as ratoon crops on a humid forest site. Significant differences between ploidy within crosses were observed for most traits. The tetraploids were taller, had longer production cycles but a shorter fruit filling time, and heavier bunches than the diploids. There were significant differences between clones of the same ploidy within crosses. Hence, individual selection within ploidy will be effective for the improvement of plantain. Most of the bunch and fruit traits were positively and significantly associated, especially at the tetraploid level. Selection for large fruit at the diploid and tetraploid levels may lead to heavy fruits, thereby resulting in hybrids bearing heavy bunches. Significant and positive correlations between plant height and bunch weight at the tetraploid level suggest that selection of dwarf plantains with heavy bunches may be difficult.

Effect of the bs1 gene in plantain-banana hybrids on response to black sigatoka

Use of resistant host genotypes is an important component of an integrated approach to control black sigatoka, a disease caused by the fungus Mycosphaerella fijiensis Morelet. The objective of the present research was to determine the role of the major gene for black sigatoka resistance (bs 1 ) in the host response to this disease. Euploid hybrids with a known genotype for the bs 1 locus were derived from triploid-diploid crosses of two French plantains and a diploid wild banana, and were assessed for their host response to black sigatoka in plant and ratoon crops in the humid forest zone of Nigeria. Host response was determined at flowering by recording the number of standing leaves, the youngest leaf with symptoms, the youngest leaf spotted, the total leaf area attacked by black sigatoka, and an index of the leaves spotted. An analysis of frequency distribution in each segregating population showed that almost all the traits displayed a normal distribution across ploidy level. This suggests that additive gene action plays an important role in the host-plant response to the fungus. Heritability, repeatibility, and intraclass correlations were calculated. The environment and the genotype-by-environment interaction significantly affected the host response to black sigatoka, which explains the low repeatibility of all traits. The intrafamily variation was larger than the interfamily variation, and most of the genetic variation in each family depended on the individual genotypes, regardless of their ploidy. The additive effect of, and the intralocus interaction at, the bs 1 locus on host response to black sigatoka were established by a one-way analysis of variance and regression analyses. Intralocus interaction in the bs 1 locus apparently regulates the appearance of symptoms on the leaf surface, whereas the additive effect and the intralocus interaction of the bs 1 locus affect disease development in the host plant. Therefore, the gene action(s) at the bs 1 locus may provide durable resistance to black sigatoka by slowing down disease development in the host plant.

A nuclear compatibility gene, Ncc-tmp, of Triticum timopheevi for the cytoplasm of Aegilops squarrosa.

A nuclear genome of timopheevi wheat is compatible with the cytoplasm of Aegilops squarrosa, while that of durum wheat is incompatible, thus no euploid hybrids with this cytoplasm could be obtained. A euploid nucleus-cytoplasm (NC) hybrid of Triticum durum cv. Langdon was produced by introgressing a NC compatibility gene (Ncc-tmp) of T. timopheevi. Southern blot analysis, using one RAPD marker (S13830) that is tightly linked to Ncc-tmp and DNAs from a series of D genome chromosome substitution lines of Langdon, demonstrated that Ncc-tmp locates on chromosome 1A. A similar function of Ncc-tmp and Ncc-sqr of Ae. squarrosa for its own cytoplasm suggested that they are homoeoallelic. Homologous sequences of two other linked RAPD markers (R03820 and R19600) were detected in all accessions of T. urartu but not in T. boeoticum. The result showed the conservation of sequences adjacent to Ncc-tmp in T. urartu, an A genome donor for emmer and timopheevi wheat. An analysis of γ-gliadin showed that the NC hybrid possesses one allele that might have been introgressed from chromosome 1G of T. timopheevi. Together with this result, 3:1 segregation of plump and shriveled seeds in BC1 of the cross between (sqr)-T. timopheevi and T. turgidum suggested the presence of an additional and functional homoeoallelic Ncc-tmp in T. timopheevi.

Effect of the black sigatoka resistance locus bs 1 and ploidy level on fruit and bunch traits of plantain-banana hybrids.

Plantain (Musa spp., AAB group) cultivation is threatened by black sigatoka, an airborne fungal (Mycosphaerella fijiensis Morelet) leaf spot disease. Several traits in plantains and bananas are mainly affected by major genes. The host resistance response to black sigatoka is under the control of at least three different genes, one major recessive gene bs1 and two other independent additive alleles. Diploid and tetraploid plantain hybrids were evaluated for bunch weight, fruit weight, fruit length and fruit circumference. The F1 euploid hybrids were derived from interspecific crosses between the resistant diploid wild banana ‘Calcutta 4’ and the susceptible triploid plantain cultivars ‘Obino l'Ewai’ and ‘Bobby Tannap’. Linear and multiple regression models, coefficients of determination, and Durbin-Watson statistics were used to determine the single and combined effects of the major locus for black sigatoka resistance and ploidy on the different traits in the progenies. Differences in yield were mainly due to changes in weight and girth of fruit, which are affected by black sigatoka disease. The combined effect of ploidy and resistance to black sigatoka was partially responsible for the quantitative trait variation in yield. As a result of the gene interaction in the black sigatoka resistance locus bs1, the partially resistant and less susceptible phenotypes showed higher yield than their more susceptible full sibs.

Effect of the parthenocarpy gene P1 and ploidy on fruit and bunch traits of plantain-banana hybrids

Plantain and banana (Musa spp. AAB and AAA groups) are perennial giant herbs of the tropics which develop parthenocarpic fruits. At least three independent but complementary dominant genes control vegetative parthenocarpy in Musa. One of these genes, P1 segregates in euploid hybrid progenies derived from crosses between triploid ‘French’ plantains and a wild nonedible diploid banana. Linear correlation and regression analyses revealed that bunch weight and fruit weight and size were positively influenced by both ploidy increases and change of recessive to dominant alleles at the P1 locus. Moreover, significant multiple regression models, including ploidy and number of copies of the P1 allele as independent variables, accounted for most of the phenotypic variation for bunch and fruit traits. The coefficients of determination of the multiple regression analyses were always smaller than estimates of broad-sense heritability for each trait. This implies that ploidy and the effect of allele substitution did not explain all the genetic variation for bunch and fruit traits. Hence, other genetic factors may explain the remaining portion of genetic variation. The potential for indirect marker-assisted selection in the seedling nursery, through the utilization of predictive multiple regression equations, was assessed by the Durbin-Watson test of residuals. The adoption of this breeding method requires the identification of DNA markers linked to the P1 gene and reliable and rapid methods to determine ploidy in seedlings

Morphological and cytological characteristics of some wheat x barley hybrids

As initial step in the transfer of dwarf bunt resistance from barley into wheat, the two cereal crops were hybridized. Using the wheat cultivars 'Fukuhokomugi' and 'Chinese Spring' (AABBDD genomes) as female parents and barley cultivar 'Luther' (II genome) as male, we synthesized 9 euploid hybrids (2n = 4x = 28; ABDI genomes). The hybrids were vigorous, but highly sterile. Meiotic analyses of seven hybrids showed considerable variation in chromosome pairing. Of the hybrids involving 'Fukuhokomugi' 3 had high pairing with a mean of 5.08-6.72 chiasmata per cell, while others had 2.16-3.52 chiasmata per cell. As many as 12 bivalents in some pollen mother cells would suggest at least some pairing between wheat and barley chromosomes. This level of homoeologous pairing, coupled with some, albeit low, female fertility of the F1 hybrids, could offer an opportunity for intergeneric gene transfers from barley into wheat and vice versa.

Factors Influencing Seed Set in Triploid Musa spp. L. and Production of Euploid Hybrids

Seed set in triploid Musa spp. L. is very low due to high levels of sterility. This makes cross breeding of plantain and banana difficult. Nonetheless, several triploid plantain and banana cultivars produce seeds after hand pollination with diploid parents. This paper reports results about the pattern of seed variation, success of in vitro germination and hybrid production after triploid-diploid crosses. Between-and within-clones differences in seed set were observed. ABB cooking bananas had the highest rates of seed production followed by AAB French plantains. Plantains produced more diploid than tetraploid offsprings after triploid-diploid crosses, which suggests that meiosis resulted in the production of balanced haploid gametes (n = x) due to normal chromosome behaviour between homologous genomes. Seed set variation within clones was influenced by both the production of 2n (= 3x) gametes in the triploid parent and climatic factors. Indeed, seed production, embryo and seed germination success, and euploid and tetraploid hybrid production followed a pattern of seasonal variation reaching a peak in September. High frequency of seed with tetraploid embryos was obtained in the period from January to mid-March. The results demonstrate that production of desirable tetraploids would be more efficient when hand pollinations are done under high temperature, high solar radiation and low relative humidity. These seem to be the most convenient conditions for relatively high production of 2n eggs as compared to haploid gametes.

Enhanced meiotic chromosome pairing in intergeneric hybrids between Triticum aestivum and diploid Inner Mongolian Agropyron

Triticum aestivum cv. Chinese Spring (2n = 6x = 42, ABD genomes) was crossed with diploid Inner Mongolian Agropyron Gaertn. species A. cristatum and A. mongolicum and reciprocal hybrids between them (2n = 2x = 14, P genome, with or without B chromosomes). Intergeneric hybrids with 2n = 27, 28, 32, and 33 chromosomes were produced by the aid of embryo rescue. The extra chromosomes in two hybrids were assumed to be B chromosomes transmitted by the male Agropyron parent. Average meiotic pairing in the euploid hybrid with 28 chromosomes was 14.38 univalents + 4.92 bivalents + 1.26 trivalents. This level of pairing higher than expected was likely due to homeologous associations between wheat chromosomes. This data indicates that the P genome of diploid as well as tetraploid Agropyron originating from Inner Mongolia possess a genetic system interfering with 5B homoeologous restricting system of wheat.Key words: intergeneric hybrids, Triticum aestivum, diploid Agropyron species, chromosome pairing.

Intergeneric hybrids between Hordeum vulgare and Elymus trachycaulus resistant to Russian wheat aphid

Intergeneric hybrids between cultivated barley (Hordeum vulgare L.) (2n = 2x = 14) and slender wheatgrass (Elymus trachycaulus (Link) Gould ex Shinners), PI 531691 (2n = 4x = 28), were produced. This slender wheatgrass accession carries resistance to Russian wheat aphid, Diuraphis noxia Mordvilko, and was used as pollen parent. The seven hybrid plants were also resistant to Russian wheat aphid. The hybrids were produced at a frequency of 0.21% of emasculated and hand-pollinated florets. Chromosome pairing in all the euploid hybrids was very low (average chiasma frequency per cell, 0.30). This indicates that the I genome from H. vulgare is remotely related to the SH genomes of E. trachycaulus. Expression of resistance to Russian wheat aphid in all the hybrid plants demonstrates that the nuclear gene or genes for insect resistance contributed by E. trachycaulus are either dominant or hemizygous effective. Five of the seven hybrid plants were vigorous and produced well-developed flowering spikes; however, they did not shed viable pollen. Since all the vegetatively cloned plants of these hybrids exhibited a high degree of perennial characteristics and could be induced to flower under a favourable light and temperature regime, they appear congenial for chromosome doubling and backcrossing. Preliminary observations suggest that successful backcrossing could be achieved using the F1 hybrids as pistillate parent and H. vulgare as pollen parent.Key words: Russian wheat aphid resistance, intergeneric hybrids, genomic relationship, chromosome pairing, Hordeum vulgare, Elymus trachycaulus.

Elimination and duplication of particular Hordeum vulgare chromosomes in aneuploid interspecific Hordeum hybrids

Seeds formed in crosses Hordeum lechleri (6x) x H. vulgare (2x and 4x), H. arizonicum (6x) x H. v. (2x), H. parodii (6x) x H. v. (2x), and H. tetraploidum (4x) x H. v. (2x) produced plants at high or rather high frequencies through embryo rescue. Giemsa C-banding patterns were used to analyze chromosomal constitutions and chromosomal locations on the methaphase plate. Among 100 plants obtained from H. vulgare (2x) crosses, 32 plants were aneuploid with 2n=29 (1), 28 (3), 27 (13), 26 (5), 25 (4), 24 (4), or 22 (2); 50 were euploid (12 analyzed), and 18 were polyhaploid (5 analyzed). Four plants had two sectors differing in chromosome number. Two of four hybrids with H. vulgare (4x) were euploid and two were aneuploid. Parental genomes were concentrically arranged with that of H. vulgare always found closest to the metaphase centre. Many plants showed a certain level of intraplant variation in chromosome numbers. Except for one H. vulgare (4x) hybrids, this variation was restricted to peripherally located non-H. vulgare genomes. This may reflect a less firm attachment of the chromosomes from these genomes to the spindle. Interplant variation in chromosome numbers was due to the permanent elimination or, far less common, duplication of the centrally located H. vulgare chromosomes in all 34 aneuploids, and in a few also to loss/gain of non-H, vulgare chromosomes. This selective elimination of chromosomes of the centrally located genome contrasts conditions found in diploid interspecific hybrids, which eliminate the peripherally located genome. The difference is attributed to changed "genomic ratios'. Derivatives of various H. vulgare lines were differently distributed among euploid hybrids, aneuploids, and polyhaploids. Chromosomal constitutions of hypoploid hybrids revealed a preferential elimination of H. vulgare chromosomes 1, 5, 6, and 7, but did not support the idea that H. vulgare chromosomes should be lost in a specific order. H. vulgare SAT-chromosomes 6 and 7 showed nucleolar dominance. Aneuploidy is ascribed to the same chromosome elimination mechanism that produces haploids in cross-combinations with H. vulgare (2x). The findings have implications for the utilization of interspecific Hordeum hybrids.

Chromosome pairing and synaptonemal complex formation in wheat-rye hybrids

The effect of chromosome 5B on chromosome pairing and chiasma formation in hybrids between allohexaploid wheat and diploid rye has been analyzed by ultrastructural investigations of synaptonemal complexes in spread silver stained microsporocytes of hybrids nullisomic for chromosome 5B, euploid hybrids and hybrids carrying an isochromosome for the long arm of chromosome 5B. At metaphase I, there were a mean number of 6.9 chiasmata per cell in the nullisomic 5B hybrids and 0.7 chiasmata per cell in the euploid hybrids, while the monoisosomic 5BL hybrids were virtually achiasmatic, with the exception of the isochromosomes where chiasmata formed regularly between the homologous/identical arms. Up to 40–50% of the complement paired with a synaptonemal complex in the three genotypes, giving rise to multiple associations often involving nearly all lateral components. In the nullisomic 5B hybrids the number of pairing partner exchanges per lateral component engaged in synapsis at zygotene was twice that found in the two other genotypes. It is concluded that nullisomy for chromosome 5B in wheat-rye hybrids results both in a lower stringency of synapsis and in permissiveness for crossing over between homoeologues.

Gene dosage effects in human diploid and tetraploid fibroblasts

The effects of cell ploidy on the biochemical characteristics of cultured cells were compared using human diploid vs tetraploid fibroblasts isolated with a non-selective method. Their DNA replication was compared by thymidine incorporation, and DNA content by Feulgen staining and quantitative analysis. Their RNA and protein content, cell sizes and the specific activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were assayed quantitatively. With the exception of RNA content, all other parameters demonstrated a 2-fold increase reflecting the increase in cell ploidy. These direct gene dosage effects on the genetic material and functional expression of the human genome were in contrast to previous observations in other species and validate the use of human intraspecific euploid hybrids for biochemical and genetic studies.

The replicative life spans of euploid hybrids derived from short-lived and long-lived human skin fibroblast cultures.

Two recent technical advances facilitate the derivation of proliferating hybrids from human diploid fibroblast strains without recourse to biochemical selection: (1) a new chemically-mediated method of somatic cell fusion (PEG-DMSO) yields hybrids at rates as high as 1 in 160 colonies after dilute plating of treated cell mixtures, and (2) a simple technology for assessment of DNA content (flow microfluorometry) permits rapid and highly sensitive monitoring of ploidy. Employing these techniques, we isolated 43 chromosomally stable hybrid clones from 12 crosses between seven different strains representing a wide range of longevities. Crosses between short-lived strains resulted in short-lived hybrid offspring, whereas hybrids derived from long-lived parents tended to be long-lived. Crosses between strains of contrasting longevities gave clones with intermediate growth potentials relative to the other types of hybrids. The failure to observe complementation (enhanced longevity) of hybrids argues against random recessive single-copy gene mutations as important determinants of clonal senescence.