A rapid method by flow cytometry for estimating persistence of tetraploid perennial ryegrass in pasture mixtures with diploid perennial ryegrass

A simple and rapid method for the estimation of the proportion of diploid and tetraploid perennial ryegrass in a mixed sward is described, based on the measurement of the nuclear DNA content of the leaf tissue cells by flow cytometry. The error of estimation was less than 5 %.     

Chromosome association and fertility in tetraploid ryegrass

In colchicine treated diploid ryegrass,Lolium perenne L. (2n=14), in addition to normal diploids, tetraploids and mixoploids, cytologically aberrant plants were observed. The latter included one aneuploid (2n=25), four translocation heterozygotes (diploid and mixoploid), and one mixoploid desynaptic plant which was a chimera of normal and desynaptic tissues as shown by successive clonal propagations. The normal diploids had chromosome association of 7 II, with an average chiasmata frequency of 12 per pollen mother cell and had 85.6 per cent pollen fertility. The tetraploids showed a mean chromosome association of 0.88 I, 4.83 II, 0.29 III and 3.91 IV, with an average chiasmata frequency of 25 per microsporocyte, and had 83.8 per cent fertile pollen. In the tetraploids, pollen fertility was negatively correlated with quadrivalent frequency and positively so with bivalent and trivalent frequencies, there being no significant correlation with univalent frequency. Among the most frequent types of quadrivalents, only the alternate chain quadrivalent frequency was positively correlated (r=+.9297) with the plllen fertility. On this basis, an increased pollen fertility in the succeeding generations of tetraploids could result from a decrease in the frequency of quadrivalent types other than the alternate chains.     

RFLP variation in diploid and tetraploid alfalfa

Summary Alfalfa (Medicago sativa L.) is a major forage crop throughout the world. Although alfalfa has many desirable traits, continued breeding is required to incorporate pest resistances and other traits. We conducted this study to determine the amount of restriction fragment length polymorphism (RFLP) variability present within and between diploid and tetraploid alfalfa populations, and whether or not this variability is sufficient for construction of an RFLP map. Diploid plants from M. sativa ssp. falcata, ssp. coerulea, and ssp. sativa and tetraploid spp. sativa cultivars Apollo, Florida 77, and Spredor 2 were included. A total of 19 cDNA clones was probed onto genomic Southern blots containing DNA digested by EcoRI, HindIII, or BamHI. Phylogenetic trees were produced, based on parsimony analysis of shared restriction fragments. Evidence for extensive gene duplication was found; most probes detected complex patterns of restriction fragments. Large amounts of variation are present within all diploid subspecies. M. sativa ssp. falcata plants formed clusters distinct from ssp. sativa or ssp. coerulea plants, which were not distinctly clustered. Some M. sativa ssp. falcata plants were more similar to the other groups than to other plants within ssp. falcata. Variation among tetraploid cultivars showed that Florida 77 and Apollo had more similarities than either showed with Spredor 2. All three cultivars showed large within-population variation, with Apollo being the most diverse and Spredor 2 the least. Based on these results, development of an RFLP map at the diploid level appears possible. Also, differentiation of cultivars, particularly ones of divergent origin, seems possible based on RFLP patterns.     

Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were isolated from several diploid and tetraploid cultivars of perennial ryegrass (Lolium perenne L.) by three different purification protocols. The apparent K_m values for substrate CO₂ were essentially identical for the fully $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$ activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report (M. K. Garrett, 1978, Nature (London)274, 913–915), these results indicate that increased ploidy level (i.e., nuclear gene dosage) has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/ oxygenase in perennial ryegrass.     

Hybrids between tetraploid Italian and perennial ryegrass

Summary Hybridization frequency was investigated between tetraploid perennial and Italian ryegrass (Lolium perenne X multiflorum) without emasculation by using genetic markers. The Italian phenotypes, fluorescentroots and awned florets, were dominant. About 82% of the plants in perennial X Italian and nearly 93 % of the plants in the reciprocal crosses were hybrids. The hybrids had a high multivalent frequency and involved homoeologous chromosome pairing. Aneuploids with 2n = 26, 27 and 29 chromosomes were present. The hybrids were highly fertile. The cytogenetic behaviour of these allopolyploids suggested that the genomes of the parental species have undergone little repatterning and have free genetic exchanges. The species maintained their self-incompatibility and cross-compatibility at the tetraploid level.     

Isozyme gene duplication in diploid and tetraploid potatoes

Summary Isozyme techniques allow the study of gene redundancy in different ploidy levels of potato (Solanum tuberosum). In tetraploid potatoes all isozyme loci are duplicated. No sign of structural or regulatory divergence was found, as is expected due to their tetrasomic inheritance patterns. In addition to this genetic redundancy, produced by a relatively recent polyploidization event, some additional redundancy was found for at least three enzymes even in diploid groups and species. These older duplicate genes show structural and regulatory divergence, indicating they appeared by a separate polyploidization event far in the past. Their common origin is still recognizable by both their expression in the same subcellular compartment and by the dimerizing ability of the isozymes they encode. To account for the present chromosome number x = 12 of the Solanaceae family, the most frequently found among the species, a hypothetical polyploidization event is proposed.     

Call degradation in diploid and tetraploid green toads

According to the 'environmental selection' hypothesis, the physical characteristics of environments influence the evolution of long-range acoustic signals by favouring those properties that decrease sound attenuation and distortion with distance. Different environments could favour different acoustic properties and therefore contribute to the intra- and interspecific variation of calls. In the present paper, we investigate whether this hypothesis could explain the differences between the advertisement calls of three taxa of central-Asian green toads: lowland diploids, highland tetraploids and lowland tetraploids. The pattern of propagation of 12 natural calls (four for each taxon) was analysed in nine localities of Kyrgyzstan and Kazakstan. We broadcast the calls and recorded them along a trasect at distances of 2, 4, 8, 16, and 32 m from the speaker, to estimate sound attenuation and propagation. Attenuation was quantified from the oscillogram (by directly calculating the SPL of calls) and from the power spectrum (by measuring the relative amplitude of the fundamental frequency) , whereas degradation was estimated by cross-correlating spectrograms of the same call at different distances. Results show that: (1) the pattern of sound propagation significantly differs among localities in relation to the different vegetation and environmental noise; (2) in most localities, call attenuation and degradation differ significantly among the three taxa ; (3) such differences are not consistent to those expected under the hypothesis of environmental selection: independent of altitude, lowland tetraploid calls fare worse than both diploid and highland tetraploid calls, whereas diploid and highland tetraploid calls show different patterns of propagation in a few localities only.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 11–26.     

Correspondence of trichome mutations in diploid and tetraploid cottons

Quantitative variation for leaf trichome number is observed within and among Gossypium species, varying from glabrous to densely pubescent phenotypes. Moreover, economically important cotton lint fibers are modified trichomes. Earlier studies have mapped quantitative trait loci (QTLs) affecting leaf pubescence in Gossypium using allotetraploids. In this study, we mapped genes responsible for leaf trichome density in a diploid A genome cross. We were able to map 3 QTLs affecting leaf pubescence based on trichome counts obtained from young leaves (YL) and mature leaves (ML). When the F(2) progeny were classified as pubescent versus glabrous, their ratio did not deviate significantly from a 3:1 model, suggesting that glabrousness is inherited in a simple Mendelian fashion. The glabrous mutation mapped to linkage group A3 at the position of major QTL YL1 and ML1 and appeared orthologous to the t1 locus of the allotetraploids. Interestingly, a fiber mutation, sma-4(ha), observed in the same F(2) population cosegregated with the glabrous marker, which indicates either close linkage or common genetic control of lint fiber and leaf trichomes. Studies of A genome diploids may help to clarify the genetic control of trichomes and fiber in both diploid and tetraploid cottons.     

Triploid and tetraploid hybrids from diploid x tetraploid crosses in Grindella (compositae)

Crosses between the diploids G. oxylepis var. eligulata Steyermark (Mexico) and G. havardii Steyermark (New Mexico) and the tetraploid G. aphanactis Rydb. (New Mexico) were made. With G. aphanactis as the pistillate parent and G. havardii as the pollen parent a triploid hybrid was obtained in which the maximum meiotic configuration observed was 6m. The plant was 10 % fertile. Two triploid hybrid plants were also obtained when G. aphanactis was used as the pistillate parent and G. oxylepis var. eligulata was the pollen parent. One plant was about 20 % fertile and the other had a maximum configuration of 3_II + 4_III. The reciprocal cross produced a tetraploid which had a maximum configuration of 6_II + 3_IV and was 8 % fertile. The tetraploid plant undoubtedly resulted from the union of an unreduced gamete from the 2n parent and a normally reduced gamete from the tetraploid. Morphology of the hybrids was usually intermediate when compared with the parental species, although some characters in the triploids were those of the diploid parent. Chromosome end arrangements of the respective genomes and putative pairing relationships are presented and phylogenetic implications are discussed. It is concluded that G. aphanactis is more closely related to G. havardii than to G. oxylepis var. eligulata.     

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.     

Pollen release by diploid and tetraploid sugar-beet plants

Pollen released during three summers at Broom's Barn, Suffolk, by diploid and tetraploid multigerm sugar-beet plants was caught by automatic volumetric spore traps and by glass funnels situated in the crop. Tetraploids produced on average 66% as much pollen as diploids, their pollen grains were larger and they needed drier air to release pollen, the concentration of which increased more slowly early in the day and was particularly small on dull, damp days. Pollen catches from diploids and tetraploids decreased at a similar rate with increasing distance from the pollinators. The germination of fruits harvested from male-sterile plants among the pollinators was consistently less with a tetraploid than with a diploid pollinator, but was not affected by distance of the male-sterile plants from either pollinator.     

Meiotic prophase in diploid and tetraploid strains ofAllomyces macrogynus

Summary Three-dimensional serial section reconstructions of meiotic prophase nuclei ofAllomyces macrogynus (Chytridiomycetes, Blastocladiales) have been carried out. Serial section reconstructions of pachytene nuclei have revealed that the fungus when grown at 23 °C is an autotetraploid and is a diploid when grown at 35 °C for at least 6 months or on a substrate containing para-fluorophenylalanine for 1–2 weeks.Studies of the duplication and migration of the centrioles during the first stages of prophase revealed the existence of four centrioles in the 23 °C strain after centriole duplication and two centrioles in the 35 °C strain after duplication. It is observed that a bivalent attaches to the nuclear envelope at a site where a centriole is situated. It is proposed that the presence of the four centrioles in the 23 °C strain is due to the fact that each bivalent is represented twice in the strain.     

A comparison of mutation induction in diploid and tetraploid rice

Summary A comparative assessment of the frequency and spectrum of chlorophyll mutations in the M ₂ and M ₃ of three diploids and one tetraploid of rice after X-irradiation was made. As well as a linear relationship of the frequency of mutations with the dose of mutagen, a saturation effect was also evident. Among the three diploids, the maximum frequency of mutations was observed in T. N. 1, followed by G. E. B. 24 and A. S. D. 8. The diploid of G. E. B. 24 showed a higher frequency of mutations than the tetraploid when measured on the M₁ plant basis only. The diploids showed a higher rate of mutations in the M ₂ than in the M₃.There was no relationship between the frequency of different types of chlorophyll mutations and the dose of X-rays. Albina, occurred in greater proportion than the other types of mutations in the diploids as well as in the tetraploid. The diploid showed a wider spectrum in the M ₂, whereas the tetraploid recorded the maximum types of mutations in the M ₃. Striking differences in the spectrum and the relative frequency of each type were observed among the three diploids and also between the diploid G. E. B. 24 and its tetraploid.The frequency and spectrum of induced mutations in the diploid and autotetraploid provided an insight into the genetic behaviour of the diploid and autotetraploid of G. E. B. 24, indicating that the genetics of diploidisation of the existing diploid rice may give evidence on the nature of the evolutionary pathway.     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

Estimation of preferential pairing in tetraploid x diploid hybridizations

Summary Crosses made between tetraploid and diploid, 2n pollen-producing species directly transfer from one-half to the entire diploid genome from the diploid to the tetraploid level, depending on the mechanism of 2n pollen formation and the amount of crossing-over that occurs. Tetraploid plants that result from tetraploid x diploid hybridizations can be further utilized in a breeding program. It is postulated that preferential pairing between homologous chromosomes derived from the original tetraploid or diploid parent occurs in the tetraploid x diploid hybrid. Depending on the genetic divergence of the species involved, preferential pairing of homologous chromosomes may range from zero to one. Theoretical estimates of the amount of preferential pairing and the standard errors of these estimates are derived for cases where the diploid parent produces 2n gametes by either a first division or a second division restitution mechanism.     

Apomixis and sexuality in diploid and tetraploid accessions of Brachiaria decumbens

 Meiotic and aposporous embryo sacs and the initial steps of parthenogenetic embryogenesis and endosperm formation were investigated in diploid and tetraploid accessions of Brachiaria decumbens in two environments, differing mainly in day length: early summer and late autumn. Both diploid and tetraploid accessions were facultative apomicts. Di(ha)ploids showed a much lower level of apomixis (10% to15%) than tetraploids (80% to 95%). No obligate sexual diploids were found; thus, their occurrence in natural populations is obscure. It is suggested that reproduction in B. decumbens, as in other agamic complexes of the Paniceae tribe, in general, approximates a diploid-tetraploid-(di)haploid reproductive cycle which does not involve triploids. The dihaploids were fertile and survived in nature. Development of the reproductive structures depended on the environment. In autumn, in contrast to early summer, many meiotic and aposporous embryo sacs degenerated during development, leading to a significant reduction in the proportion of parthenogenetic embryos. Whether this effect can be attributed to day length or simply to age remains to be investigated. The ratio of aposporous to sexual embryo sacs was relatively stable over the two seasons. Received: 15 April 1998 / Revision accepted: 13 October 1998