Two independent loci control agamospermy (Apomixis) in the triploid flowering plant Erigeron annuus

Asexual seed production (agamospermy) via gametophytic apomixis in flowering plants typically involves the formation of an unreduced megagametophyte (via apospory or diplospory) and the parthenogenetic development of the unreduced egg cell into an embryo. Agamospermy is almost exclusively restricted to polyploids. In this study, the genetic basis of agamospermy was investigated in a segregating population of 130 F(1)'s from a cross between triploid (2n = 27) agamospermous Erigeron annuus and sexual diploid (2n = 18) E. strigosus. Correlations between markers and phenotypes and linkage analysis were performed on 387 segregating amplified fragment length polymorphisms (AFLPs). Results show that four closely linked markers with polysomic inheritance are significantly associated with parthenogenesis and that 11 cosegregating markers with univalent inheritance are completely associated with diplospory. This indicates that diplospory and parthenogenesis are unlinked and inherited independently. Further, the absence of agamospermy in diploid F(1)'s appears to be best explained by a combination of recessive-lethal gametophytic selection against the parthenogenetic locus and univalent inheritance of the region bearing diplospory. These results may have major implications for attempts to manipulate agamospermy for agricultural purposes and for interpreting the evolution of the trait.     

Inheritance of apomeiosis (diplospory) in fleabanes (Erigeron, Asteraceae)

Unreduced egg formation (apomeiosis) in flowering plants is rare except when it is coupled with parthenogenesis to yield gametophytic apomixis via apospory or diplospory. Results from genetic mapping studies in diverse apomictic taxa suggest that apomeiosis and parthenogenesis are genetically linked, a finding that is compatible with the conventional rationale that apomeiosis is unlikely to evolve independently because of deleterious fitness consequences. An Erigeron annuus (apomictic) x E. strigosus (sexual) genetic mapping population, however, included a high proportion of plants that were highly apomeiotic (diplosporous) but nonapomictic; that is, they lacked autonomous seed production. To evaluate the function and inheritance of diplospory in Erigeron, a diplosporous triploid (2n=3x=27) seed parent was crossed with a sexual diploid (2n=2x=18) E. strigosus pollen parent to produce an F1 of 31 plants. Chromosome numbers and molecular markers (AFLPs) document the inheritance of the maternal genome through unreduced eggs resulting in recombinant but predominantly (77%) tetraploid F1s (2n=4x=36; 2n+n, B(III)). Quantitative evaluation shows continuous variation in the proportion of diplosporous (vs meiotic) ovules (41-89%) in tetraploid F1s despite the presumed equal genetic contribution from the diplosporous mother. These findings demonstrate the functional independence of diplospory and suggest that variation in the trait in F1s is likely due to segregating paternal modifiers. In addition, of six aneuploid (4x-1, 4x-2) F1s, three lack a subset of maternal AFLP markers. These plants likely arose from aberrant megagametogenesis resulting in the loss of maternal chromatin prior to fertilization.     

Apomixis via recombination of genome regions for apomeiosis (diplospory) and parthenogenesis in Erigeron (daisy fleabane, Asteraceae)

Apomixis in daisy fleabanes (Erigeron annuus and E. strigosus) is controlled by two genetically unlinked loci that regulate, independently, the formation of unreduced female gametophytes (apomeiosis, diplospory) and autonomous seed formation (parthenogenesis). In this work, fully apomictic F2s were regenerated by crossing F1s bearing, separately, these two functional regions. Two triploid (3x = 2n = 27) highly diplosporous F1s served as seed parents to an aneuploid (2x + 1 = 2n = 19) meiotic pollen donor bearing four AFLP markers linked to parthenogenetic seed formation but producing only abortive embryos and endosperm. Of 408 hybrids, 21 (5.1%) produced seed. Nine of these putative apomicts were tetraploids (4x), likely combining an unreduced egg from the diplosporous seed parent and a haploid gamete from the pollen parent (3x + x). The other 12 hybrid apomicts were pentaploid, interpreted as arising from the fusion of an unreduced diplosporous egg with an unreduced sperm cell (3x + 2x). Analysis indicated that all but three of the 21 synthetic apomicts recombined markers linked to diplospory and parthenogenesis. In addition, three additional hybrids combined markers linked to the two functional regions but produced only aborted embryos. The apomicts varied in percentage of diplosporous ovules (4.7–95.3% of all ovules produced) and in percentage of ovules that developed into seed (3.8–58.0%). These results support the hypothesis that apomeiosis and autonomous seed formation are genetically distinct, and that the traits can be separated and recombined to create hybrids exhibiting apomixis at near wildtype levels.     

Mendelian segregation for two-factor apomixis in Erigeron annuus (Asteraceae)

The inheritance of asexual seed development (apomixis) in Erigeron annuus (Asteraceae) was evaluated in a triploid (2n=3x=27) population resulting from a cross between an apomictic tetraploid (2n=4x=36) pollen parent and a sexual diploid (2n=2x=18) seed parent. Diplospory (unreduced female gametophyte formation) and autonomous development (embryo and endosperm together) segregated independently in the population yielding four distinct phenotype classes: (1) apomictic plants combining diplospory and autonomous development, (2) diplosporous plants lacking autonomous development, (3) meiotic plants with autonomous (though abortive) development and (4) meiotic plants lacking autonomous development. Each class was represented by approximately one-quarter of the population (n=117), thus corresponding to a two-factor genetic model with no linkage (chi(2)=2.59, P=0.11). Observations demonstrate that autonomous embryo and endosperm development (jointly) may occur in either reduced or unreduced egg cells. The cosegregation of the traits is attributed to tight linkage or pleiotropy. The data are consistent with the hypothesis that autonomous development in E. annuus is regulated by a single fertilization factor, F, which initiates development of both the embryo and the endosperm in the absence of fertilization.     

Formation of unreduced megaspores (diplospory) in apomictic dandelions (Taraxacum officinale, s.l.) is controlled by a sex-specific dominant locus

In apomictic dandelions, Taraxacum officinale, unreduced megaspores are formed via a modified meiotic division (diplospory). The genetic basis of diplospory was investigated in a triploid (3x = 24) mapping population of 61 individuals that segregated approximately 1:1 for diplospory and meiotic reduction. This population was created by crossing a sexual diploid (2x = 16) with a tetraploid diplosporous pollen donor (4x = 32) that was derived from a triploid apomict. Six different inheritance models for diplospory were tested. The segregation ratio and the tight association with specific alleles at the microsatellite loci MSTA53 and MSTA78 strongly suggest that diplospory is controlled by a dominant allele D on a locus, which we have named DIPLOSPOROUS (DIP). Diplosporous plants have a simplex genotype, Ddd or Dddd. MSTA53 and MSTA78 were weakly linked to the 18S-25S rDNA locus. The D-linked allele of MSTA78 was absent in a hypotriploid (2n = 3x - 1) that also lacked one of the satellite chromosomes. Together these results suggest that DIP is located on the satellite chromosome. DIP is female specific, as unreduced gametes are not formed during male meiosis. Furthermore, DIP does not affect parthenogenesis, implying that several independently segregating genes control apomixis in dandelions.     

The occurrence of phenotypically complementary apomixis-recombinants in crosses between sexual and apomictic dandelions (<Emphasis Type="Italic">Taraxacum officinale</Emphasis>)

Apomictic seed development in dandelion ( Taraxacum officinale) involves (1) restitutional meiosis (diplospory), (2) egg cell parthenogenesis, and (3) autonomous endosperm development. The question is whether these elements of apomixis are controlled by one single gene or by several independent genes. Five triploid non-apomictic hybrids, obtained in diploid sexual × triploid apomict crosses were characterized using cyto-embryological and genetic methods. Nomarski-differential interference contrast microscopy and the transmission of microsatellite markers and ploidy levels indicated that the hybrids combined elements of the apomictic and the sexual developmental pathway. Hybrids form two complementary groups with respect to the presence or absence of parthenogenesis and autonomous endosperm development. The occurrence of complementary apomixis-recombinants suggests that parthenogenesis and autonomous endosperm development in Taraxacum are regulated independently by different genes. This study also indicates that early embryo development is independent of endosperm formation, but that endosperm is essential for later embryo growth.     

Automictic reproduction in interspecific hybrids of poeciliid fish

Automixis, the process whereby the fusion of meiotic products restores the diploid state of the egg, is a common mode of reproduction in plants but has also been described in invertebrate animals. In vertebrates, however, automixis has so far only been discussed as one of several explanations for isolated cases of facultative parthenogenesis. Analyzing oocyte formation in F1 hybrids derived from Poecilia mexicana limantouri and P. latipinna crosses (the cross that led to the formation of the gynogenetic Poecilia formosa), we found molecular evidence for automictic oocyte production. The mechanism involves the random fusion of meiotic products after the second meiotic division. The fertilization of diploid oocytes gives rise to fully viable triploid offspring. Although the automictic production of diploid oocytes as seen in these F1 hybrids clearly represents a preadaptation to parthenogenetic reproduction, it is also a powerful intrinsic postzygotic isolation mechanism because the resulting next generation triploids were always sterile. The mechanism described here can explain facultative parthenogenesis, as well as varying ploidy levels reported in different animal groups. Most importantly, at least some of the reported cases of triploidy in humans can now be traced back to automixis.     

Pattern of variation of agamospermous Limonium (Plumbaginaceae) in the British Isles

An investigation of cytological, palynological, and taxometric variation in Limonium binervosum (G. E. Sm.) C. E. Salmon has revealed an extensive pattern of hierarchical relationships within this agamospermous group in the British Isles. The group is either triploid (2n = 27) or aneuploid tetraploid (usually 2n = 35) with a highly jumbled karyotype. Pollen is either not produced or of very low stainability (in the range 0–38%). Well stained pollen grains are usually abnormally large resulting from a failure of a meiotic division. These facts as well as the presence of only a single self-incompatible polledstigma morph in the majority of the group suggest that it is obligately agamospermic. The very strong correlation of taxometric relationship and geographical distance between variants suggests that evolution in the group has occurred by the gradual accumulation of mutations giving rise to morphologically and geographically related seed clones. The hierarchical pattern of variation as revealed by cluster analysis has been used as the basis of a hierarchical classification of the group.     

远缘杂交形成的二倍体鱼和多倍体鱼生殖细胞染色体研究

本文采用性腺染色体制片及组织学切片方法,系统地研究了不同发育时期的鲫鲤杂交第二代(F2) (2n=100)、异源四倍体鲫鲤(4n=200)、三倍体鲫鱼(3n=150))、雌核发育二倍体鲫鲤第二代(G2)(2n=100)及鲤鱼(Cypninus carpio L)(2n=100)(对照组)生殖细胞的染色体特征.研究结果表明,对照组中鲤鱼精原细胞染色体数与体细胞染色体数一致,为二倍体精原细胞(2n=100),而远缘杂交形成的二倍体鱼和多倍体鱼的生殖细胞中则观察到明显的染色体数加倍现象,其中,鲫鲤杂交第二代(F2)精巢生殖细胞染色体数加倍现象特别丰富,占检测的染色体分裂相的21.6%,为其产生不减半的二倍体配子提供了直接的细胞学证据,同时也说明远缘杂交是导致生殖细胞染色体数加倍的一个重要因素.该研究在探讨多倍体鱼的发生及鱼类遗传育种方面具有重要意义.     

三倍体短葶飞蓬的发现及其在育种上的意义

对云南省野生居群的短葶飞蓬有丝分裂的细胞学特征进行观察研究。在云南省大理苍山、腾冲杨家坪的二倍体野生居群中,发现有三倍体个体的存在。苍山居群的两种核型分别为2n=2x=18=4m 10sm 4st,2n=3x=27=6m 12sm 9st;杨家坪居群的两种核型分别为2n=2x=18=6m 10sm 2st,2n=3x=27=3m 15sm 9st。通过对其生物学特性及表型的分析,认为短葶飞蓬可以采取多倍体育种方式获得更优良的品种。     

The Significance of Agamospermous Triploid Pollen Donors in the Sexual Relationships between Diploids and Triploids in Taraxacum (Compositae)

Abstract We review in this article the investigations of the significance of agamospermous triploid pollen donors in the sexual relationships between diploids and triploids in Taraxacum . Crossing experiments between diploid sexual mother plants and agamospermous polyploid pollen donors and recent isozyme analyses of the progeny have exhibited the following results: 1) Pollen from Agamospermous polyploid pollen donors have the potential to give rise to the polyploid agamospermous offspring when fertilizing diploid sexual plants. Ploidy level of the progeny is usually the same or higher, but occasionally lower, compared to the pollen donor. 2) Diploid progeny also occur from diploid (♀)-polyploid (♂) crosses, however, these diploids were in our results not hybrids but the results of self-fertilization of the diploids. The self-fertilization is regarded as a cosequence of the breakdown of the self-incompatibility barrier through the sterile triploid's pollen. This breakdown is in all probability a common phenomenon in diploid (♀)-polyploid (♂) crosses. Some examples suggest that agamospermous polyploids can increase their genetic diversity through obtaining genes from coexisting diploids. The evolutionary implications of this phenomenon and the reduction mechanism of chromosome number through agamospermous pollengenesis are discussed.     

Quantitative variation for apomictic reproduction in the genus Boechera (Brassicaceae)

? Premise of the study: The evolution of asexual seed production (apomixis) from sexual relatives is a great enigma of plant biology. The genus Boechera is ideal for studying apomixis because of its close relation to Arabidopsis and the occurrence of sexual and apomictic species at low ploidy levels (diploid and triploid). Apomixis is characterized by three components: unreduced embryo-sac formation (apomeiosis), fertilization-independent embryogenesis (parthenogenesis), and functional endosperm formation (pseudogamy or autonomous endosperm formation). Understanding the variation in these traits within and between species has been hindered by the laborious histological analyses required to analyze large numbers of samples. ? Methods: To quantify variability for the different components of apomictic seed development, we developed a high-throughput flow cytometric seed screen technique to measure embryo:endosperm ploidy in over 22000 single seeds derived from 71 accessions of diploid and triploid Boechera. ? Key results: Three interrelated features were identified within and among Boechera species: (1) variation for most traits associated with apomictic seed formation, (2) three levels of apomeiosis expression (low, high, obligate), and (3) correlations between apomeiosis and parthenogenesis/pseudogamy. ? Conclusions: The data presented here provide a framework for choosing specific genotypes for correlations with large "omics" data sets being collected for Boechera to study population structure, gene flow, and evolution of specific traits. We hypothesize that low levels of apomeiosis represent an ancestral condition of Boechera, whereas high apomeiosis levels may have been induced by global gene regulatory changes associated with hybridization.     

Analysis of meiosis in triticale (XTriticosecale Wittmack) X rye (Secale cereale L.) F1 hybrids at three ploidy levels

Summary Triticales (XTriticosecale Wittmack) at three ploidy levels (8x, 6x, 4x, x=7) were crossed with diploid rye (Secale cereale L.) to produce a solitary hypopentaploid hybrid (2n=32), and a number of tetraploid (2n=4x=28) and triploid (2n=3x=21) hybrids. The hybrids exhibited a morphology which was intermediate between the parents. The number of bivalents ranged from 1–7 (4.65 per cell) in hypopentaploid, from 2–12 (7.13 per cell) in tetraploid and from 4–9 (6.84 per cell) in triploid hybrids. In 4x and 3x hybrids, trivalents and quadrivalents were also observed at low frequencies (range 0–1; mean 0.01–0.03 per cell). Chiasmata frequency was highest in triploid hybrids (12.44 per cell), lowest in hypopentaploid (5.37 per cell) and intermediate in tetraploids (10.54 per cell). More than 7¹¹ were found in 39.7% pollen mother cells (PMC's) in the 4x hybrids and in 5.0% PMCs in 3x hybrids. It is concluded that an increase in the relative proportion of wheat chromosomes in the hybrids had a slight suppression effect on homologous as well as homoeologous pairing of rye chromosomes. Contrary to this, the relative increase in rye complement promoted homoeologous pairing between wheat chromosomes. In triploid hybrids, the chiasmata frequency as well as the c value were the highest, suggesting that in tetraploid hybrids rye chromosomes had a reduced pairing (low frequency of ring bivalents).     

Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male

Diploid and triploid interspecific hybrid male progeny obtained from mating Misgurnus anguillicaudatus with M. mizoleis were reported to have histologically fertile and sterile testes, respectively. However, their reproductive capacity is still unclear because mating tests have not been examined using mature hybrids. Here, we examined physiological and genetic characteristics of spermatozoa of diploid and triploid hybrids. In diploid hybrid males, 1n, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only 1n spermatozoa of diploid hybrid males was fertile to produce larva. The chromosomes of diploid hybrid males were transmitted to spermatozoa by random segregation between the homologous chromosomes because most larvae had one allele derived from both M. anguillicaudatus and M. mizolepis at all loci examined. In triploid hybrid males, spermatozoa could be categorized to three different types based on their ploidy status. Type 1: In the first and second males, sperm samples mainly comprised 6n spermatozoa. Motility and fertility were not recorded. Type 2: The third male gave a large proportion of 6n spermatozoa as well as a small proportion of 1n spermatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only 1n spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such 1n spermatozoa, normal larvae hatched. 1n spermatozoa of the triploid hybrid male only included the M. anguillicaudatus genome. In Misgurnus fishes, diploid hybrid males exhibited semi-sterility or slight fertility. On the contrary, triploid hybrid males were sometime fertile due to the production of 1n spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.     

Interspecific hybrids between a transgenic rapeseed (Brassica napus) and related species: cytogenetical characterization and detection of the transgene.

In interspecific hybrids produced between a transgenic rapeseed, an allotetraploid species, resistant to herbicide, phosphinotricin, and five diploid related species, the risk for gene introgression in weed genomes was explored through cytogenetic and bar gene characterizations. Among the 75 hybrids studied, most had the expected triploid structure, with the exception of B. napus - B. oleracea amphidiploid plants and one B. napus - S. arvensis amphidiploid plant. In triploid hybrid plants, the reciprocal hybrids did not exhibit any difference in their meiotic behavior. The comparison of the percentage of chromosome pairing in the hybrids with that of haploid rapeseed permit to conclude that allosyndesis between AC genomes and related species genomes took place. This possibility of recombination was confirmed by the presence of multivalent associations in all the interspecific hybrids. Nevertheless, in B. napus - B. adpressa hybrids a control of chromosome pairing seemed to exist. The possibility of amphidiploid plant production directly obtained in the F1 generation increased the risk of gene dispersal. The B. napus - B. oleracea amphidiploid plant presented a meiotic behavior more regular than that of the B. napus - S. arvensis amphidiploid plant. Concerning the herbicide bar gene characterization, the presence of the gene detected by DNA amplification was correlated with herbicide resistance, except for two plants. Different hypotheses were proposed to explain these results. A classification of the diploid species was established regarding their gene dispersal risk based on the rate of allosyndesis between chromosomes of AC genomes of rapeseed and the genomes of the related species.     

Genotypic variation in agamospermous Erigeron compositus (Asteraceae)

Few integrative analyses of the structure of agamospermous plant populations have been conducted. Erigeron compositus occurs in montane western North America and comprises both sexual and agamospermous populations. Sexual E. compositus has previously been characterized as outcrossing and predominantly diploid (2n = 18). Agamic E. compositus is usually hexaploid (2n = 54), though counts herein range from 2n = 36 to 2n = 80. Starch-gel electrophoresis, cytology, and analysis of pollen production were used to evaluate variation within and among agamospermous populations. Fifteen enzyme loci were used to identify 24 unique multilocus genotypes in seven populations, an average of 3.4 genotypes per population. Proportion of distinct genotypes per population sample size (GIN) and measures of genetic diversity (D) and evenness (E) are 0.10, 0.48, and 0.61, respectively, which indicate that E. compositus maintains levels of diversity similar to other agamospermous taxa. Most agamospermous populations are mosaics comprising groups of genetically distinct individuals that are frequently distinguished by cytotype and capacity for pollen production. The geographical and ecological separation of sexual and agamospermous populations make it unlikely that gene flow from sexual populations is a direct source of genetic variation in agamospermous populations. Instead, crossing between genetically distinct facultative agamosperms probably accounts for most variation. Genetic and morphological evidence document one such putative crossing event. Agamospermous E. compositus is very similar genetically to sexual E. compositus. Allozyme analysis further shows that genetic variation in agamospermous populations is partitioned among a few highly heterozygous genotypes, whereas sexual populations maintain numerous genotypes of relatively low heterozygosity.     

Chromosome numbers and pollen stainability of three species of Pacific Island breadfruit (Artocarpus, Moraceae)

Chromosome numbers were determined for 48 accessions of breadfruit (Artocarpus altilis, A. mariannensis, and A. camansi [Moraceae]) from 16 Pacific Island groups, Indonesia, and the Philippines. Artocarpus camansi and A. mariannensis exhibit counts of 2n = 56; 2n = 56 (diploidy) and 2n = 84 (triploidy) were observed for A. altilis. Most diploid cultivars of A. altilis were seeded, but two cultivars with reduced seed number were observed. Micronesian accessions included putative interspecific hybrids between A. altilis and A. mariannensis. The majority of these accessions were seedless diploids, but triploid putative hybrids were also observed. Pollen stainablility was shown to correlate with the degree of seediness.     

Reproductive isolation of a new hybrid species, Senecio eboracensis Abbott & Lowe (Asteraceae)

The nature and extent of reproductive isolation was examined between a new self-compatible hybrid species Senecio eboracensis (2n=40) and its parents, self-incompatible S. squalidus (2n=20) and self-compatible S. vulgaris (2n=40). The triploid F(1) of S. eboracensis x S. squalidus exhibited very low seed set (x=0.63%), and F(2) and F(3) progeny were able to recover nominal levels of fertility (x=23.9 and 9.7%), while F(1) and F(2) offspring of S. eboracensis x S. vulgaris showed reduced seed set (x=63.8 and 58.8%). In both cases, evidence from previous work indicates that reduced fertility is associated with meiotic chromosome mispairing, and is a likely consequence of recombining both parental genomes within this new taxon. No hybrid offspring between S. eboracensis and S. squalidus were found in the wild, and only one such hybrid was recorded among 769 progeny produced by S. eboracensis surrounded by S. squalidus on an experimental plot. Natural crossing between S. eboracensis and S. vulgaris was recorded to be very low (between 0 and 1.46%) in the wild, but rose to 18.3% when individuals of S. eboracensis were surrounded by plants of S. vulgaris. It was concluded that strong breeding barriers exist between the new hybrid species and its two parents. Prezygotic isolation between S. eboracensis and S. vulgaris is likely to be largely due to both species reproducing by predominant self-fertilisation. However, differences recorded for germination, seedling survival, time of flowering and characters associated with pollinator attraction, plus significant clumping of juvenile and adult conspecifics in the wild, probably also contribute to reproductive isolation and ecological differentiation.     

Diploid and triploid offspring of triploid agamosporous fernDryopteris pacifica

A cytological and reproductive study of the diploid and triploid agamosporousDryopteris pacifica was made to elucidate the origin of its infraspecific cytotypes. Some triploids produced 16 spore mother cells (SMCs) sometimes with n=41II+41I chromosomes, in addition to eight SMCs with n=123II, in each sporangium. In the former case the 16 SMCs usually underwent abnormal meiosis to give rise to some 50 spores, some of which were regular-shaped; in the latter the eight SMCs multiplied into 32 spores by normal meiosis. We found that spores from one of the triploid plants developed into either diploid or triploid gametophytes, which further apogamously produced diploid or triploid sporophytes, respectively. This novel mechanism of ploidy reduction is discussed in relation to the origin of diploid agamosporous ferns, the taxonomic complexity of the species, and the correlation of agamospory with polyploidy. The mechanism is also compared to that operating in agamospermous angiosperms.     

Parthenogenesis in the parasitic and free-living forms of Strongyloides papillosus (Nematoda,Rhabdiasoidea)

Both parasitic and free-living females of a calf strain of Strongyloides papillosus have a chromosome number of 2n=4. Both forms reproduce by diploid parthenogenesis. Oocytes of parasitic females undergo only one homeotypic maturation division without homologous chromosome pairing (mitotic parthenogenesis). Oocytes of free-living females show normal pairing and disjunction of the homologous chromosomes, but only one diploid polar body is expelled (meiotic parthenogenesis). Reconstitution of the diploid chromosome number occurs by separation of the two sister chromatids of each univalent during or after anaphase I.This investigation was supported by the Consiglio Nazionale delle Ricerche (C.N.R.) of Italy.