Evaluation of the effect of pollen irradiation on karyotype variability in cotton plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearragements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M1 plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M1 after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

Isolation of Deficiencies in the Arabidopsis Genome by γ-Irradiation of Pollen

Chromosomal deficiencies are a useful genetic tool in fine-scale genetic mapping and the integration of physical and visible marker genetic maps. Viable overlapping deficiencies may permit gene cloning by subtractive procedures and provide a means of analyzing the functional importance of different chromosomal regions. A method is described for isolation of deficiencies in the Arabidopsis genome which encompass specific loci and other extended chromosomal regions. The technique employs pollen mutagenized by γ-irradiation to pollinate marker lines homozygous for recessive mutations. Deficiencies at specific loci were detected by screening for marker phenotypes in the F(1). Screening for lethal mutations in the F(1)/F(2) confirmed specific deficiencies and revealed other deficiencies that did not overlap the marker loci. Further evidence for such mutations was provided by distorted F(2) segregation of the chromosomal markers linked to putative deficiencies. Maintainable (transmissible) and non-transmissible deficiencies were demonstrated by their pattern of inheritance in subsequent generations.     

Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response

Polyploidy, the inheritance of more than two genome copies per cell, has played a major role in the evolution of higher plants. Little is known about the transition from diploidy to polyploidy but in some species, triploids are thought to function as intermediates in this transition. In contrast, in other species triploidy is viewed as a block. We investigated the responses of Arabidopsis thaliana to triploidy. The role of genetic variability was tested by comparing triploids generated from crosses between Col-0, a diploid, and either a natural autotetraploid (Wa-1) or an induced tetraploid of Col-0. In this study, we demonstrate that triploids of A. thaliana are fertile, producing a swarm of different aneuploids. Propagation of the progeny of a triploid for a few generations resulted in diploid and tetraploid cohorts. This demonstrated that, in A. thaliana, triploids can readily form tetraploids and function as bridges between euploid types. Genetic analysis of recombinant inbred lines produced from a triploid identified a locus on chromosome I exhibiting allelic bias in the tetraploid lines but not in the diploid lines. Thus, genetic variation was subject to selection contingent on the final ploidy and possibly acting during the protracted aneuploid phase.     

Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses

Shoot organogenesis was induced from 2- and 6-week-old callus derived from the leaves of Arabidopsis thaliana ecotype Columbia (2n = 10). Regenerated plants were evaluated for chromosomal variations by means of flow cytometry and fluorescent in situ hybridization (FISH). Flow cytometric measurements revealed the occurrence of diploid, tetraploid, and octoploid plants among the regenerants of 2-week-old calli, whereas only diploid and tetraploid plants were regenerated from the 6-week-old calli. Chromosome counting showed that plants developed from the 2-week-old calli exhibited mixoploidy and a high frequency of aneuploid cells. These plants were infertile and displayed altered morphology. FISH with 5S and 25S rDNA probes allowed to detect some structural chromosomal rearrangements in regenerated plants. Along with cells which exhibited correct localisation of rDNA loci, also cells bearing chromosomal translocations, deletions or duplications were found. The type of structural aberrations varied between diploid and tetraploid regenerants.     

Parameters affecting telomere-mediated chromosomal truncation in Arabidopsis

Conversion of a double-strand break into a telomere is a dangerous, potentially lethal event. However, little is known about the mechanism and control of de novo telomere formation (DNTF). DNTF can be instigated by the insertion of a telomere repeat array (TRA) into the host genome, which seeds the formation of a new telomere, resulting in chromosome truncation. Such events are rare and concentrated at chromosome ends. Here, we introduce tetraploid Arabidopsis thaliana as a robust genetic model for DNTF. Transformation of a 2.6-kb TRA into tetraploid plants resulted in a DNTF efficiency of 56%, fivefold higher than in diploid plants and 50-fold higher than in human cells. DNTF events were recovered across the entire genome, indicating that genetic redundancy facilitates recovery of DNTF events. Although TRAs as short as 100 bp seeded new telomeres, these tracts were unstable unless they were extended above a 1-kb size threshold. Unexpectedly, DNTF efficiency increased in plants lacking telomerase, and DNTF rates were lower in plants null for Ku70 or Lig4, components of the nonhomologous end-joining repair pathway. We conclude that multiple competing pathways modulate DNTF, and that tetraploid Arabidopsis will be a powerful model for elucidating the molecular details of these processes.     

Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.     

Karyological characterization of sugar beet gynogenetic lines cultured in vitro

Flow cytometry was used to screen ploidy levels in 47 cultured in vitro sugar beet gynogenetic lines of various origin and age, obtained after plant regeneration from unfertilized ovules. When donor plants were diploid, the majority of regenerants were found to have cells with 1C, 2C and 4C relative DNA content (mainly haploid and diploid) and there were large differences in the rate of spontaneous in vitro chromosome doubling between individual homozygous lines. Six ovule-derived lines regenerated from fertile and sterile diploid donors of forty-five lines were solid diploids from the very early stages of their in vitro cultivation, and thus could not be classified as doubled haploids. In the case of tetraploid donor plants, the gynogenetic regenerants demonstrated 2x-ploidy level. The results obtained in chimeric plants with both haploid and diploid cells indicated the possibility to overcome mixoploidy by their re-cultivation through generative shoot tip culture. The flow cytometry method confirmed data obtained by conventional microscopic chromosome counting in dividing leaf cells and was found very useful for screening of a large number of regenerants and for characterizing the process of in vitro gynogenetic lines formation in sugar beet.     

Evaluation of the Effect of Pollen Irradiation on Karyotype Variability in M1 Cotton Plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearrangements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M₁ plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M₁ after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

A fertile amphiploid between diploid wheat (Triticum tauschii) and crested wheat grass (Agropyron cristatum).

Alloploidy, one of the most efficient evolutionary mechanisms in nature, has not been extensively exploited in plant breeding programmes. Many genomic combinations remain to be created by plant breeders, to be used directly as new crops or indirectly to widen the genetic basis of crops. The Triticeae tribe, to which wheat belongs, is among the botanical groups in which this strategy has been successfully explored. However, there remain valuable genomic combinations that have not been obtained at the diploid level. The Agropyron complex (wheat-grasses) has recently been the focus of attention for interspecific hybridization, but intergeneric hybrids or amphiploids with wheat have not been reported at the diploid level. Here we report synthesis of a tetraploid amphiploid between Triticum tauschii and Agropyron cristatum by crossing two tetraploid accessions. Using total genome in situ hybridization (GISH) staining on metaphase I pollen mother cells, data on allosyndetic and autosyndetic chromosome pairing have been obtained. These data support the view that the A. cristatum tetraploid parent used in the synthesis of the amphiploid has a segmental alloploidy nature.     

Colinearity and gene density in grass genomes

Grasses are the single most important plant family in agriculture. In the past years, comparative genetic mapping has revealed conserved gene order (colinearity) among many grass species. Recently, the first studies at gene level have demonstrated that microcolinearity of genes is less conserved: small scale rearrangements and deletions complicate the microcolinearity between closely related species, such as sorghum and maize, but also between rice and other crop plants. In spite of these problems, rice remains the model plant for grasses as there is limited useful colinearity between Arabidopsis and grasses. However, studies in rice have to be complemented by more intensive genetic work on grass species with large genomes (maize, Triticeae). Gene-rich chromosomal regions in species with large genomes, such as wheat, have a high gene density and are ideal targets for partial genome sequencing.     

Pairing Competition between Identical and Homologous Chromosomes in Autotetraploid Rye. I. Submetacentric Chromosomes

Meiotic pairing preferences between identical and homologous but not identical chromosomes were analyzed in ten induced tetraploid/diploid chimaeral rye plants (Secale cereale) heterozygous for telomeric heerochromatin C-bands in both arms of chromosome 1R. These plants were the progeny of two crosses between only one plant of cv. Petkus, used as male, and two plants of the inbred lines E and R, respectively. Different pairing preferences for chromosome 1R were found: (1) between plants, (2) between chromosome arms within the same plant and (3) between bivalents and multivalents within the same plant. The possible influence in the preferences of several factors such as differences in C-heterochromatin content in the chromosomes analyzed, specific genetic control and independence in pairing behavior between both arms and partner exchange is discussed.     

PARTHENOGENESIS IN TETRAPLOID LILIUM LONGIFLORUM

Emsweller , S. L., and Joseph Uhring . (U.S.D.A., Beltsville, Md.) Parthenogenesis in tetraploid Lilium longiflorum. Amer. Jour. Bot. 49(9): 978–984. Illus. 1962.—Nine maternal polyhaploids from 1 capsule and 1 tetraploid from another were produced following pollination of 2 tetraploid Lilium longiflorum plants with pollen from diploids of the same species. One of the 9 plants had 25 chromosomes; the extra chromosome was identified as a modified D. Two other plants had 2 new chromosomes each and the remaining 6 had 24 unmodified chromosomes. Translocations in meiosis of the tetraploid produced the new chromosomes. One plant obtained from a second capsule had 48 chromosomes. The 9 plants were smaller than diploids and the 48-chromosome plant was considered a diploid until mitosis was observed. The 9 plants originated from unfertilized eggs of the tetraploid, and the 48-chromosome plant presumably from chromosome doubling of an egg cell.     

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.     

Genome evolution among cruciferous plants: a lecture from the comparison of the genetic maps of three diploid species--Capsella rubella, Arabidopsis lyrata subsp. petraea, and A. thaliana

Comparative mapping in cruciferous plants is ongoing, and recently two additional genetic maps of diploid Capsella and Arabidopsis lyrata subsp. petraea have been presented. We compared both maps with each other using the sequence map and genomic data resources from Arabidopsis thaliana as a reference. The ancestors of the species pair Capsella-Arabidopsis diverged from one another approximately 10-14 million years ago (mya), whereas Arabidopsis thaliana and Arabidopsis lyrata have been separated since roughly 5-6 mya. Our analysis indicated that among diploid Capsella and Arabidopsis lyrata all eight genetic linkage groups are totally colinear to each other, with only two inversions significantly differentiating these two species.By minimizing the number of chromosomal rearrangements during genome evolution, we presented a model of chromosome evolution involving all three species. From this scenario, it is obvious that Arabidopsis thaliana underwent a dramatic genome reconstruction, with a base chromosome number reduction from five to eight and with approximately 1.3 chromosomal rearrangements per million years. In contrast, the terminal lineage leading to Capsella has only undergone less than 0.09 rearrangements per million years. This is the same rate as calculated for Arabidopsis lyrata since its separation from the Capsella lineage 10-14 mya. These results are in strong contrast to all overestimated rates calculated from comparisons of the systems Arabidopsis thaliana and Brassica, and our data demonstrate the problematic nature of both model systems.     

Evaluation of the effect of pollen irradiation on karyotype variability in M2 cotton plants

The karyotypes of biomorphologically abnormal cotton (Gossypium hirsutum L.) plants obtained in M2 after pollination with pollen irradiated at dose rates 10, 15, 20, and 25 Gy were studied. Various genomic and chromosomal mutations were detected in 57 M2 families. The primary monosomics isolated in M2 were found to be cytologically more stable and more viable, since they had higher meiotic indices, pollen fertility, and seed formation. In M2, a decrease in the number of plants with multiple karyotype aberrations and interchromosomal exchanges with high frequency of multivalent formation was observed. The multivalents had diverse patterns and types of chromosome segregation and translocation complexes. Their pollen fertility was higher than in translocants found in M1. Desynapsis often occurred in M2, including plants with chromosome deficiency or rearrangements. The variation in the number of univalents in various cells was found to result from different expression of synaptic genes. The results indicate stabilization of karyotypes, increase in cytologic stability and viability, and the absence of sterility in aberrant plants.     

Ecological factors influencing tetraploid establishment in snow buttercups (Ranunculus adoneus , Ranunculaceae): minority cytotype exclusion and barriers to triploid formation

Polyploid speciation is an ongoing, important source of angiosperm diversity. However, the barriers to polyploid speciation and mechanisms of establishment remain poorly understood for all but a few species. Several factors likely to have influenced tetraploid establishment, including barriers to triploid formation, consequences of mixed-ploidy pollen loads, and the reproductive success of the minority cytotype, were examined in snow buttercups (Ranunculus adoneus) through a series of pollination and transplant experiments. Tetraploid snow buttercups do not have stigmatic barriers to pollen from diploid plants, nor does pollen from tetraploid plants have an advantage over pollen from diploids when on tetraploid stigmas. Tetraploid plants transplanted into a diploid population produced 50% fewer seeds than tetraploid plants in a tetraploid population. Intrinsic barriers to triploid formation were relatively weak, but few triploid seeds formed when mixed-ploidy pollen was present. Fecundity of triploid plants was very low, and no tetraploid offspring resulted. These results indicate that in snow buttercups, a triploid plant will contribute 0.8% of the tetraploid seeds of a minority tetraploid plant making it a minor contributor to the demographics of tetraploid establishment. The reproductive costs facing minority cytotype plants may explain the previously observed spatial segregation in snow buttercups.     

Estimation of efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberration in M2 of cotton Gossipium hirsutum L

Cytogenetic analysis of M2 plants after irradiation of cotton by thermal neutrons was performed in 56 families. In 40 plants of 27 M2 families, different abnormalities of chromosome pairing were found. These abnormalities were caused by primary monosomy, chromosomal interchange, and desynapsis. The presence of chromosome aberrations in some cases decreased meiotic index and pollen fertility. Comparison of the results of cytogenetics analysis, performed in M1 and M2 after irradiation, showed a nearly two-fold decrease in the number of plants with chromosomal aberrations in M2, as well as narrowing of the spectrum of these aberrations. The latter result is explained by the fact that some mutations are impossible to detect in subsequent generations because of complete or partial sterility of aberrant M1 plants. It was established that the most efficient radiation doses for inducing chromosomal aberrations in the present study were 15 and 25 Gy, since they affected survival and fertility of altered plant to a lesser extent.     

Aneuploidy and polyploidization in haploid tissue cultures of Larix decidua

Haploid embryogenic lines of Larix decidua which had been maintained on medium free of plant growth regulatiors were screened for chromosomal abnormalities. Chromosome squashes were prepared from fast-growing suspension culture. alternatively, a method using burst protoplasts was devised for improving chromosome spreads. One line showed polyploidization with chromosome mumbers ranging from haploid to tetraploid. This line is presently predominantly diploid. Aneuploidization also occurred. The most effective method for counting chromosomes was from burst protoplasts. It is recommended that long-term cultures of conifer tissues be checked for genetic stability.