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.     

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 index, 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 multivalent configurations had different types and chromosome disjunctions. 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.     

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.     

Estimation of the efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberrations in M1 of cotton Gossypium hirsutum l

Exposure of cotton seeds to thermal neutrons at doses of 15, 25, and 35 Gy was shown to induce many altered plants, including sterile and chimeric ones. Most of these phenotypic changes were shown to result from novel genomic, chromosomal, and desynaptic mutations. The presence of these mutations in the karyotype of M, plants often decreased meiotic index and pollen fertility. In translocation forms, the decrease in pollen fertility was caused by the prevalence of quadrivalents in form of rings and chains with adjacent segregation of chromosomes from the translocation complexes. Based on the shapes and sizes of multivalent associations, we performed preliminary localization of translocation breakpoints. A specific feature of the effect of thermal neuron irradiation in M1 was induction of numerous unique chromosomal aberrations, consisting in the appearance in the same plant of several types of mutations (genomic and chromosomal), exchange complexes in the same nucleus, and multiple exchanges involving three nonhomologous chromosomes.     

Estimation of the efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberrations in M<Subscript>1</Subscript> of cotton <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.

Exposure of cotton seeds to thermal neutrons at doses of 15, 25, and 35 Gy was shown to induce many biomorphologically abnormal plants, including sterile and chimeric ones. Most of these phenotypic changes were shown to result from novel genomic, chromosomal, and desynaptic mutations. The presence of these mutations in the karyotype of M₁ plants often decreased meiotic index and pollen fertility. In translocation forms, the decrease in pollen fertility was caused by the prevalence of quadrivalents in form of rings and chains with adjacent segregation of chromosomes from the translocation complexes. Based on the shapes and sizes of multivalent associations, we performed preliminary localization of translocation breakpoints. A specific feature of the effect of thermal neuron irradiation in M₁ was induction of numerous unique chromosomal aberrations, consisting in the appearance in the same plant of several types of mutations (genomic and chromosomal), interchange complexes in the same nucleus, and multiple interchanges involving three nonhomologous chromosomes.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Estimation of efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberration in M<Subscript>2</Subscript> of cotton <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> 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 twofold 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.     

Chromosome Rearrangement by Ectopic Recombination in Drosophila Melanogaster: Genome Structure and Evolution

Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure. A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments. Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences. The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.     

Induced interchange heterozygosity in diploid Chrysanthemum

Summary Following a programme of recurrent irradiation and planned hybridization, it has been possible in Chrysanthemum carinatum to synthesise an interchange stock in which 12 of the 18 chromosomes are involved in rearrangements and form a single multiple association during meiosis. Induced interchange heterozygosity of this type has been made use of for studying the relationship between fertility and meiotic behaviour. Relatively high fertility of the interchange heterozygotes was found to be mainly due to a pronounced tendency of the interchange multiples to show preferentially a particular type of orientation at metaphase. Factors governing the orientation of multiple association resulting from interchange heterozygosity have been discussed.     

The Development and a Cytogenetic Study of Monosomics of Gossypium Hirsutum L.

Monosomics of cotton (Gossypium hirsutum L.) were obtained by irradiation of pollen by -rays and by irradiation of seeds by thermal neutrons. Many monosomics were derived directly from irradiation, but a number of monosomics were also recovered in the progeny of plants with translocations and of desynaptic plants. Only 28 primary monosomics showed normal pairing at metaphase-1 of meiosis. The others formec rare trivalents or additional univalents. Partial desynapsis was detected in some monosomics. The pollen fertility levels of monosomics are presented. New morphological characters were detected among the monosome plants of cotton.     

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.     

Cytogenetic effects of the treatment of cotton seeds with thermal neutrons

After irradiation of cotton seeds with thermal neutrons, primary and tertiary monosomics and also plants with translocations were detected among offsprings M1 and M2. The interchanges had high frequency of multivalents at metaphase-I of meiosis. Genetic analysis of chlorophyll deficient cotton mutants has shown that recessive monogenic factors caused new mutations of the types xantha and viridis. New viridis mutant was also characterized by the heterozygosity of interchromosome translocations.     

Heterochromatic regions in corn A- and B- chromosomes

A collection of maize forms from the Vavilov All-Russia Institute of Plant Breeding (VIR) was studied. We compared variation in the following traits: the number and size of heterochromatic knob regions (HKRs) of chromosomes of mother pollen cells at pachytene depending on the presence (B+) or absence (B0) of B-chromosomes; size of B-chromosomes (general, of heterochromatic part); and the frequency of B+ plants in groups of forms contrasting in the HKR number (10-15, 4-10, 2-7, 1-4). It was shown that B chromosomes had statistically significantly different effects on HKR polymorphism, relative heterochromatin "content" in the cell of multiknob and knobless forms, selection of plants for early ear flowering in these forms. The combination of maximum (and minimum) sizes of heterochromatic regions of A- and B-chromosomes was established; the genomic level of control of the trait is suggested. The role of the relationship of polymorphism at heterochromatic regions of A- and B-chromosomes between them and with the systems of maize reproduction is considered as a mechanism of maintenance of optimum plant heterozygosity via adaptive ontogenetic redistribution of heterochromatin among loci, chromosomes, and gametes.     

Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.     

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.     

An Analysis of Regional Constraints on Exchange in DROSOPHILA MELANOGASTER Using Recombination-Defective Meiotic Mutants

The frequency of crossing over per unit of physical distance varies systematically along the chromosomes of Drosophila melanogaster . The regional distribution of crossovers in a series of X chromosomes of the same genetic constitution, but having different sequences, was compared in the presence and absence of normal genetically mediated regional constraints on exchange. Recombination was examined in Drosophila melanogaster females homozygous for either normal sequence X chromosomes or any of a series of X chromosome inversions. Autosomally, these females were either (1) wild type, (2) homozygous for one of several recombination-defective meiotic mutations that attenuate the normal regional constraints on exchange or (3) heterozygous for the multiply inverted chromosome TM2. The results show that the centromere, the telomeres, the heterochromatin and the euchromatic-heterochromatic junction do not serve as elements that respond to genic determinants of the regional distribution of exchanges. Instead, the results suggest that there are several elements sparsely distributed in the X chromosome euchromatin. Together with the controlling system affected by recombination-defective meiotic mutations, these elements specify the regional distribution of exchanges. The results also demonstrate that the alteration in the distribution of crossovers caused by inversion heterozygosity (the interchromosomal effect) results from the response of a normal controlling system to an overall increase in the frequency of crossing over, rather than from a disruption of the system of regional constraints on exchange that is disrupted by meiotic mutations. The mechanisms by which regional constraints on exchange might be established are discussed, as is the possible evolutionary significance of this system.     

Recombination suppression in heterozygotes for a pericentric inversion induces the interchromosomal effect on crossovers in Arabidopsis

During meiosis, recombination ensures allelic exchanges through crossovers (COs) between the homologous chromosomes. Advances in our understanding of the rules of COs have come from studies of mutations including structural chromosomal rearrangements that, when heterozygous, are known to impair COs in various organisms. In this work, we investigate the effect of a large heterozygous pericentric inversion on male and female recombination in Arabidopsis. The inversion was discovered in the Atmcc1 mutant background and was characterized through genetic and next‐generation sequencing analysis. Reciprocal backcross populations, each consisting of over 400 individuals, obtained from the mutant and the wild type, both crossed with Landsberg erecta, were analyzed genome‐wide by 143 single‐nucleotide polymorphisms. The negative impact of inversion became evident in terms of CO loss in the rearranged chromosome in both male and female meiosis. No single‐CO event was detected within the inversion, consistent with a post‐meiotic selection operating against unbalanced gametes. Cytological analysis of chiasmata in F₁ plants confirmed that COs were reduced in male meiosis in the chromosome with inversion. Crossover suppression on the rearranged chromosome is associated with a significant increase of COs in the other chromosomes, thereby maintaining unchanged the number of COs per cell. The CO pattern observed in our study is consistent with the interchromosomal (IC) effect as first described in Drosophila. In contrast to male meiosis, in female meiosis no IC effect is visible. This may be related to the greater strength of interference that constrains the CO number in excess of the minimum value imposed by CO assurance in Arabidopsis female meiosis.     

Occurrence of aneuploid progenies from an asynaptic amphidiploid ofScilla scilloides (lindley) druce II. Mechanism of production of the various aneuploid progenies

The mechanism of production of the various aneuploid progenies was clarified in the asynaptic amphidiploid plants (2n=34+4f+2F, AABB) ofScilla scilloides. Its asynaptic nature and chromosomal stickiness lead to the unequal segregation at anaphase I (AI) in PMC's. The observed values in 18 segregation patterns, 17:17 to 0: 34, were different from the expected values estimated from random segregation of chromosomes. Nevertheless, the preferential transmission of special chromosomes among genomes A (x=8=a₁−a₈) and B (x=9=b₁−b₉) had not occurred. As the result of unequal segregation, the pollen grains with various chromosome numbers were observed. Almost all of the 200 pollen grains contained chromosome numbers more than 17 (range 8 to 34). The observed values of each chromosome number were roughly similar to the expected values of containing the complete set of genome A or B in the random distribution without preferential segregation of chromosomes at AI. The difference between the index of polien mitosis and the pollen fertility was significant in the Wilcoxon matched-pairs signed rank test and suggested the selection for some genomically unbalanced pollen grains during maturation. Consequently, viable pollen grains with various chromosome constitutions are a few (mean pollen fertility of 5.8%) but might produce many aneuploids by self- and cross-pollination.     

Short-term storage of cotton pollen

Several methods of storing cotton pollen (Gossypium hirsutum L.) were evaluated. A successful pollen storage method that maintains fertility would enhance the crossing of breeding materials. Storing pollen at ultra-low temperatures in liquid nitrogen or at 5°C was not successful. No storage method maintained pollen fertility for more than 72 h. Cotton pollen did maintain adequate fertility up to 24 h at 10 and 15°C, at both low and high humidity when the pollen was stored in the detached flowers. Minimally acceptable pollen fertility was maintained in flowers stored at 15°C at 100% R.H. for 72 h. Use of these methods will allow for better utilization of parental plants when both parents do not flower on the same days.USDA-ARS, in cooperation with the New Mexico Agricultural Experimental Station, Journal Article No. 1161, Las Cruces, NM 88003, USA     

Neutron- and X-ray-induced mutations at the yellow, white, forked and vermilion loci of Drosophila melanogaster; a preliminary analysis

Neutrons and X-rays were used to induce mutations at the yellow, white, vermilion and forked loci of Drosophila melanogaster by irradiation of spermatozoa in males. The mutations were characterized for the presence and location of simultaneously induced rearrangements and recessive lethal mutations. F1 females carrying induced visible mutations were identified, described and tested for fertility. The data are given in this paper. The proportions of mutants at the 4 loci, the ratios of whole-body: mosaic mutations, and the fertility of the mutant-carrying F1 females were similar for both types of radiation. Differences were observed between the frequencies of induced visible mutations and the rates of coincident visible and lethal induction. Although the analysis of the mutant chromosomes has not yet been completed, our data can be interpreted as providing confirmation that there are qualitative differences between the genetic effects of neutrons and X-rays.