Characterization of a partial wheat-Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. A wheat-Thinopyrum intermedium partial amphiploid, TAI8335, and its alien parent were characterized by a combination of genomic in situ hybridization (GISH) and cytological observations. Evidence from GISH indicated that the donor parent Th. intermedium possessed seven pairs of S, seven J(s) and 21 J chromosomes. Mitotic observation showed that the majority of TAI8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential GISH patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium DNA as probes revealed that TAI8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of J(s) - and one pair of J-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved J and S genome. Two of the telochromosomes in the aneuploid plants originated from the J genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid TAI8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

C-banding and fluorescence in situ hybridization studies of the wheat-alien hybrid 'Agrotana'.

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat x Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' x 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat-alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat-alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.     

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.     

Molecular cytogenetic characterization of an alloplasmic durum wheat line with a portion of chromosome 1D of Triticum aestivum carrying the scs(ae) gene.

Triticum aestivum (2n = 6x = 42, AABBDD) with Triticum longissimum (2n = 2x = 14; S1S1) cytoplasm ((lo) cytoplasm) has normal fertility and plant vigor. However, the nucleus of durum wheat (Triticum turgidum (2n = 4x = 28, AABB)) is incompatible with the T. longissimum cytoplasm, producing non-viable progeny. This incompatibility is alleviated by scs(ae), a species cytoplasm-specific (scs) gene, on the long arm of chromosome 1D (1DL) of common wheat. The hemizygous (lo) durum scs(ae) line is male sterile and is maintained by crossing to normal durum wheat. After pollination, the seeds produced are either plump and viable (with scs(ae)) or shriveled and inviable (without scsae). Thus, the chromosome with scs(ae) is inherited as a whole without recombination. The objectives of this study were to characterize the chromosome carrying scs(ae) and to determine the process through which this gene was introgressed into the (lo) durum background. Molecular marker analysis with 27 probes and primers mapped to homoeologous group 1 and genomic in situ hybridization using differentially labeled total genomic DNA of durum wheat and Aegilops tauschii suggest the presence of a 1AL segment in place of the distal region of 1DL. Owing to the absence of any detectable duplications or deletions, homoeologous recombination is the most likely mechanism by which this introgression occurred.     

Construction and molecular and cytogenetic analyses of euploid (2n = 42) and telocentric addition (2n = 42 + 2t) alloplasmic lines (Hordeum marinum subsp gussoneanum)-Triticum aestivum

Individual plants from the BC1F5 and BC1F6 backcross progenies of barley--wheat (= H. geniculatum All.) (2n = 28) x T. aestivum L. (2n = 42)] and the BC1F6 progeny of their amphiploids were used to obtain alloplasmic euploid (2n = 42) lines L-28, L-29, and L-49 and alloplasmic telocentric addition (2n = 42 + 2t) lines L-37, L-38, and L-50. The lines were examined by genomic in situ hybridization (GISH), microsatellite analysis, chromosome C-banding, and PCR analysis of the mitochondrial 18S/5S repeat. Lines L-29 and L-49 were characterized by substitution of wild barley chromosome 7H1 for common wheat chromosome 7D. In line L-49, common wheat chromosomes 1B, 5D, and 7D were substituted with homeologous barley chromosomes. Lines L-37, L-38, and L-50 each contained a pair of telocentric chromosomes, which corresponded to barley chromosome arm 7H'L. All lines displayed heteroplasmy for the mitochondrial 18S/5S locus; i.e., both barley and wheat sequences were found.     

Molecular characterization of a wheat--Thinopyrum ponticum partial amphiploid and its derivatives for resistance to leaf rust.

Leaf rust (caused by Puccinia triticina Eriks.) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang has provided several leaf rust resistance genes to protect wheat from this fungal disease. Three chromosome substitution lines, Ji806, Ji807, and Ji859, and two chromosome addition lines, Ji791 and Ji924, with a winter growing habit were developed from crosses between wheat (Triticum aestivum L. em Thell.) and the wheat - Th. ponticum partial amphiploid line 693. These lines were resistant to leaf rust isolates from China. Sequence-tagged site (STS) analysis with the J09-STS marker, which is linked to the gene Lr24, revealed that the partial amphiploid line 693 and all of the substitution and addition lines carried gene Lr24. Genomic in situ hybridization (GISH) analysis was carried out on chromosome preparations using total genomic DNA from Pseudoroegneria strigosa (M. Bieb) A. L?ve (St genome, 2n = 14) as a probe in the presence of total genomic DNA from T. aestivum 'Chinese Spring' wheat (ABD genomes, 2n = 42). The GISH analysis demonstrated that these lines had a pair of chromosomes displaying the typical pattern of a Js genome chromosome. This indicates that the chromosome that carries gene Lr24 belonged to the Js genome of Th. ponticum. In addition to 40 wheat chromosomes, eight Js and eight J genome chromosomes were also differentiated by GISH in the partial amphiploid line 693. Since most sources of Lr24 have a red grain color, the white-colored seeds in all of these substitution and addition lines, together with high protein content in some of the lines, make them very useful as a donor source for winter wheat breeding programs.     

Rust resistance in Triticum cylindricum Ces. (4x, CCDD) and its transfer into durum and hexaploid wheats.

In order to counteract the effects of the mutant genes in races of leaf rust (Puccinia recondita f.sp. tritici Rob. ex Desm.) and stem rust (P. graminis f.sp. tritici Eriks. &Henn.) in wheat, exploration of new resistance genes in wheat relatives is necessary. Three accessions of Triticum cylindricum Ces. (4x, CCDD), Acy1, Acy9, and Acy11, were tested with 10 races each of leaf rust and stem rust. They were resistant to all races tested. Viable F1 plants were produced from the crosses of the T. cylindricum accessions as males with susceptible MP and Chinese Spring ph1b hexaploid wheats (T. aestivum, 6x, AABBDD), but not with susceptible Kubanka durum wheat (T. turgidum var. durum, 4x, AABB), even with embryo rescue. In these crosses the D genome of hexaploid wheat may play a critical role in eliminating the barriers for species isolation during hybrid seed development. The T. cylindricum rust resistance was expressed in the F1 hybrids with hexaploid wheat. However, only the cross MP/Acy1 was successfully backcrossed to another susceptible hexaploid wheat, LMPG-6. In the BC2F2 of the cross MP/Acy1//LMPG-6/3/MP, monosomic or disomic addition lines with resistance to either leaf rust race 15 (infection types (IT) 1=, 1, or 1+; addition line 1) or stem rust race 15B-1 (IT 1 or 1+; addition line 2) were selected. Rust tests and examination of chromosome pairing of the F1 hybrids and the progeny of the disomic addition lines confirmed that the genes for rust resistance were located on the added T. cylindricum C-genome chromosomes rather than on the D-genome chromosomes. The T. cylindricum chromosome in addition line 2 was determined to be chromosome 4C through the detection of RFLPs among the genomes using a set of homoeologous group-specific wheat cDNA probes. Addition line 1 was resistant to the 10 races of leaf rust and addition line 2 was resistant to the 10 races of stem rust, as was the T. cylindricum parent. The added C-genome chromosomes occasionally paired with hexaploid wheat chromosomes. Translocation lines with rust resistance (2n = 21 II) may be obtained in the self-pollinated progeny of the addition lines through spontaneous recombination of the C-genome chromosomes and wheat chromosomes. Such translocation lines with resistance against a wide spectrum of rust races should be potentially valuable in breeding wheat for rust resistance.     

Characterization of a Wheat- wheatgrass Translocation Line by FISH

The genomic DNA of wheatgrass (Agropyron intermedium (Host) P.B. = Elytrigia intermedia (Host) Nevski = Thinopyrum intermedium (Host) Barkworth and Dewey) was labelled with biotin-16-dUTP as a probe, and genomic DNA of common wheat ( Triticum aestivum L. ) "Chinese Spring" was used for blocking. Wheat-wheatgrass line 33 was examined by fluorescence in situ hybridization (FISH) technique. The terminal regions of a pair of chromosomes showed green fluorescent signals. It has been concluded that chromosome segrnents containing alien genes of wheatgrass are located at the terminal regions of wheat chromosomes in wheat-wheatgrass line 33, and the translocated segments were small. Wheat-wheatgrass line 33 has been proved to be a translocation line with chromosome segments of wheatgrass translocated to the terminal regions of wheat chromoSomes.     

Synthesis and cytological characterization of trigeneric hybrids of durum wheat with and without Ph1.

Wild grasses in the tribe Triticeae, some in the primary or secondary gene pool of wheat, are excellent reservoirs of genes for superior agronomic traits, including resistance to various diseases. Thus, the diploid wheatgrasses Thinopyrum bessarabicum (Savul. and Rayss) A. Love (2n = 2x = 14; JJ genome) and Lophopyrum elongatum (Host) A. Love (2n = 2x = 14; EE genome) are important sources of genes for disease resistance, e.g., Fusarium head blight resistance that may be transferred to wheat. By crossing fertile amphidiploids (2n = 4x = 28; JJEE) developed from F1 hybrids of the 2 diploid species with appropriate genetic stocks of durum wheat, we synthesized trigeneric hybrids (2n = 4x = 28; ABJE) incorporating both the J and E genomes of the grass species with the durum genomes A and B. Trigeneric hybrids with and without the homoeologous-pairing suppressor gene, Ph1, were produced. In the absence of Ph1, the chances of genetic recombination between chromosomes of the 2 useful grass genomes (JE) and those of the durum genomes (AB) would be enhanced. Meiotic chromosome pairing was studied using both conventional staining and fluorescent genomic in situ hybridization (fl-GISH). As expected, the Ph1-intergeneric hybrids showed low chromosome pairing (23.86% of the complement), whereas the trigenerics with ph1b (49.49%) and those with their chromosome 5B replaced by 5D (49.09%) showed much higher pairing. The absence of Ph1 allowed pairing and, hence, genetic recombination between homoeologous chromosomes. Fl-GISH analysis afforded an excellent tool for studying the specificity of chromosome pairing: wheat with grass, wheat with wheat, or grass with grass. In the trigeneric hybrids that lacked chromosome 5B, and hence lacked the Ph1 gene, the wheat-grass pairing was elevated, i.e., 2.6 chiasmata per cell, a welcome feature from the breeding standpoint. Using Langdon 5D(5B) disomic substitution for making trigeneric hybrids should promote homoeologous pairing between durum and grass chromosomes and hence accelerate alien gene transfer into the durum genomes.     

Production of durum wheat substitution haploids from durum x maize crosses and their cytological characterization.

The objective of this study was to investigate the effect of individual durum wheat (Triticum turgidum L.) chromosomes on crossability with maize (Zea mays L.) and to cytologically characterize the haploids recovered. Fourteen 'Langdon' (LDN) D-genome disomic substitution lines, a LDN Ph mutant (Ph1b ph1b), and normal 'Langdon' were pollinated with maize pollen. After pollination, hormonal treatment was given daily for up to 14 days. Haploid embryos were obtained from all lines and were aseptically cultured. From a total of 55,358 pollinated florets, 895 embryos were obtained. Only 14 of the embryos germinated and developed into healthy plants. Different substitution lines showed varying degrees of success. The most successful was the substitution 5D(5B) for both embryo formation and haploid plantlet production. These results indicate that the substitution of 5D for 5B confers on durum wheat a greater ability to produce haploids. Fluorescent genomic in situ hybridization (GISH) showed that the substitution haploids consisted of 7 A-genome chromosomes, 6 B-genome chromosomes, and 1 D-genome chromosome. Triticum urartu Turn. genomic DNA was efficient in probing the 7 A-genome chromosomes, although the D-genome chromosome also showed intermediate hybridization. This shows a close affinity between the A genome and D genome. We also elucidated the evolutionary translocation involving the chromosomes 4A and 7B that occurred at the time of evolution of durum wheat. We found that the distal segment translocated from chromosome 7B constitutes about 24% of the long arm of 4A.     

Production,morphology, and cytogenetic analysis of Elymus caninus (Agropyron caninum) x Triticum aestivum F1 hybrids and backcross-1 derivatives

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD) and wheatgrass, Etymus caninus (Agropyron caninum) (2n=4x=28, SSHH) — the first successful report of this cross. Reciprocal crosses and genotypes differed for percent seed set, seed development and F₁ hybrid plant production. With E. caninus as the pollen parent, there was no hybrid seed set. In the reciprocal cross, seed set was 23.1–25.4% depending upon wheat genotype used. Hybrid plants were produced only by rescuing embryos 12–13 days post pollination with cv Chinese Spring as the wheat parent. Kinetin in the medium facilitated embryo germination but inhibited root development and seedling growth. The hybrids were vigorous, self sterile, and intermediate between parents. These had expected chromosome number (2n=5x=35, ABDSH), very little chromosome pairing (0.51 II, 0.04 III) and some secondary associations. The hybrids were successfully backcrossed with wheat. Chromosome number in the BC₁ derivatives varied 54–58 with 56 as the modal class. The BC₁ derivatives showed unusually high number of rod bivalents or reduced pairing of wheat homologues. These were sterile and BC₂ seed was produced using wheat pollen.     

Barley chromosome identification using genomic in situ hybridization in the genome of backcrossed progeny of barley-wheat amphiploids [H. geniculatum All. (2n = 28) x T. aestivum L. (2n = 42)] (2n = 70)

Genomic in situ hybridization (GISH) has been used to study characteristics of the formation of alloplasmic lines detected among self-pollinated backcrossed progeny (BC1F5-BC1F8) of barley--wheat amphiploids [Hordeum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42)] (2n = 70). The chromosome material of the wild barley H. geniculatum has been shown to contribute to these lines. For example, fifth-generation plants (BC1F5) had genotypes (2n = 42w + 2g), (2n = 42w + 1g + 1tg), and (2n = 41w + 1g), where w is common wheat chromosomes, g is barley (H. geniculatum) chromosomes, and tg is the telocentric chromosome of wild barley. Beginning from the BC1F6 generation, alloplasmic telocentric addition lines (2n = 42 + 2tg) and (2n = 42 + 1tg) appear. This lines has been found cytogenetically unstable. The progeny of each of these cytological types include not only the (2n = 42 + 2tg) and (2n = 42 + 2tg) addition plants, but also plants with the monosomic (2n = 41 + 1tg) and the disomic (2n = 40 + 2tg) substitutions, as well as the (2n = 41 + 2tg) plants, which lack one wheat chromosome and have two telocentric barley chromosomes. It has been demonstrated that the selection for well-filled grains favors the segregation of telocentric addition lines (2n = 42 = 2tg) and (2n = = 42 + 1tg).     

Genetic characterization of androgenic progeny derived from Lolium perenne x Festuca pratensis cultivars

Androgenesis and genetic characterization of androgenic progeny from Lolium perenne x Festuca pratensis cultivars (2n = 4x = 28) were investigated in order to develop a novel grass that combines favourable attributes of parent plants. A successful androgenesis was obtained using PG-96 medium. The green plant regeneration was 46%, 35% and 17% for Bx350, Bx351 and Prior, respectively, and over 800 green plants have been obtained. Androgenic progeny showed a large variation in freezing tolerance, 7% of 292 progeny exceeding that of freezing hardy F. pratensis despite containing chromosomes of L. perenne. More than 60% of 175 flowering progeny produced dehiscent anthers with pollens. Androgenic plants contained 14 or 28 chromosomes. There were 188 (56%), 204 (77%) and 114 (81%) dihaploids from Bx350, Bx351 and Prior, respectively. The nuclear DNA content varied significantly even between plants with the same chromosome number. High levels of chromosome pairing and recombination were observed by genomic in situ hybridization analysis because of close homology between two genomes. The results indicated that androgenic progeny of Festulolium showed high level of genetic variation, and provide potential for accelerating selection efficiency.     

Cytogenetics of Triticum x Dasypyrum hybrids and derived lines

Genomic in situ hybridization was used to study Triticum x Dasypyrum wide hybrids and derived lines. A cytogenetic investigation was carried out in progenies of (i) amphiploids derived from T. turgidum var. durum (T. durum; 2n = 14; genomes AABB) x D. villosum (2n = 14; genome VV), (ii) three-parental hybrids (T. durum x D. villosum) x T. aestivum (2n = 42, genomes A'A'B'B'D'D'), and (iii) T. aestivum aneuploid lines carrying D. villosum chromosomes or chromatin. The amphiploids derived from T. durum x D. villosum showed a stable chromosomal constitution, made up of 14 V chromosomes, 14 chromosomes carrying the wheat A genome and 14 chromosomes carrying the B genome. High karyological instability was observed in the progenies of three-parental hybrids ([T. durum x D. villosum] x T. aestivum). Plants having the expected 14 A chromosomes, 14 B chromosomes, 7 D chromosomes, and 7 V chromosomes were rather rare (4.5%). Many progeny plants (45.5%) had the hexaploid wheat genome with 42 chromosomes and lacked any detectable D. villosum chromatin. Other plants (50%) had 14 A chromosomes and 14 B chromosomes, plus variable numbers of D and V chromosomes, the former being better retained than the latter in most cases. Some T. aestivum lines carrying D. villosum chromosomes or chromatin, as the result of addition, substitution, or recombination events or even a combination of these karyological events, were found to be stable. Other lines were unstable, and these lines carried 1V, 3V, or 5V chromosomes or their portions. Substitution or recombination events where 1V chromosomes were involved could concern the homeologous counterparts in both the A and B and D genomes of wheat. No line could be recovered where the shorter arm of 3V chromosomes was present. Changes in the morphology and banding pattern of V chromosomes were observed in hybrids that did not carry the entire D. villosum complement. By comparing the results of our cytogenetic analyses with certain phenotypic characteristics of the lines studied, genes for discrete traits could be assigned to specific V chromosomes or V chromosome arms. From the frequency of V chromosomes that were involved in chromatin exchanges with or substituted for one of their homeologous counterparts in the A, B, and D wheat genomes, it was inferred that D. villosum belongs to the same phyletic lineage as T. urartu (donor of the A genome of wheat) and Aegilops speltoides (B genome), and that Ae. squarrosa (D genome) diverged earlier from D. villosum.     

Identification of mitotic chromosomes of tuberous and non-tuberous solanum species (Solanum tuberosum and Solanum brevidens) by GISH in their interspecific hybrids.

GISH (genomic in situ hybridization) was applied for the analysis of mitotic chromosome constitutions of somatic hybrids and their derivatives between dihaploid clones of cultivated potato (Solanum tuberosum L.) (2n = 2x = 24, AA genome) and the diploid, non-tuberous, wild species Solanum brevidens Phil. (2n = 2x = 24, EE genome). Of the primary somatic hybrids, both tetraploid (2n = 4x) and hexaploid (2n = 6x) plants were found with the genomic constitutions of AAEE and AAEEEE, respectively. Androgenic haploids (somatohaploids) derived from the tetraploid somatic hybrids had the genomic constitutions of AE (2n = 2x = 24) and haploids originating from the hexaploid hybrids were triploid AEE (2n = 3x = 33 and 2n = 3x = 36). As a result of subsequent somatic hybridization from a fusion between dihaploid S. tuberosum (2n = 2x = 24, genome AA) and a triploid somatohaploid (2n = 3x = 33, genome AEE), second-generation somatic hybrids were obtained. These somatic hybrids were pentaploids (2n = 5x, genome AAAEE), but had variable chromosome numbers. GISH analysis revealed that both primary and second-generation somatic hybrids had lost more chromosomes of S. brevidens than of S. tuberosum.     

Chromosome sorting in tetraploid wheat and its potential for genome analysis

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.     

Molecular cytogenetic analysis of intergeneric chromosomal translocations between wheat (Triticum aestivum L.) and Dasypyrum villosum arising from tissue culture.

Fluorescence in situ hybridization (FISH) was applied with total genomic DNA extracted from Dasypyrum villosum (L.) Candargy as a probe to characterize chromosome translocations arising from tissue culture in hybrids of Triticum aestivum x (T. durum - D. villosum, amphiploid). Chromosome translocations between wheat and D. villosum occurred in callus cells at an average frequency of 1.9%. Translocations existed not only in callus cells but also in regenerants. Three plants with translocation chromosomes were characterized among 66 regenerants of T. aestivum 'Chinese Spring' x 'TH1W' and 'NPFP' x 'TH1'. One of them proved to be a reciprocal translocation with an exchange of about one third of a wheat chromosome arm with about one half of a chromosome arm of D. villosum. The breakpoints of the other two translocations were located at, or near centromeres. The results are similar for both callus cells and regenerants and provide further evidence that translocations take place in tissue culture. Other structural chromosomal changes, for example, fragments, telocentrics, dicentromeres, and deletions, as well as numerical alterations including aneuploidy and polyploidy were recorded both in callus cells and regenerants.     

Production and identification of primary trisomics in diploid Agropyron cristatum (crested wheatgrass).

Six of the seven possible primary trisomics in Agropyron cristatum were produced. Based on morphology, arm length ratios, and C-banding patterns, they were identified as primary trisomics for chromosomes A, B, C, D, E, and G. Agropyron cristatum is one of several species constituting the crested wheatgrass complex. All species in this complex contain one basic genome (P). A study was conducted to produce and identify a primary trisomic series that will be used to map genes to individual chromosomes. A population of 157 plants were generated by crossing autotriploids (PPP) with diploid (PP) A. cristatum: 58 were diploid (2n = 14), 76 were primary trisomies (2n = 15), 17 were double trisomic (2n = 16), 4 were triple trisomics (2n = 14 + 3), 1 was telocentric trisomic (2n = 14 + 1 telo), and 1 was tetratrisomic (2n = 14 + 4). Karyotype analysis of acetoorcein-stained chromosomes was carried out using the CHROMPAC III computer program; for analysis of C-banded karyotypes, the computer imaging analysis program PCAS (Plant Chromosome Analysis System) was used to identify the primary trisomics. Of the 47 primary trisomics analyzed, 21 plants had one extra satellited chromosome E, 18 with the satellited D chromosome, 3 each for chromosomes B and G, and 1 each for chromosomes C and A. Chromosome pairing was studied in trisomies B, D, E, and G. Trisomics for chromosomes B and G were similar in their mieotic behavior. Each had a trivalent frequency of about 60% and pollen stainability of less than 40%. Trisomics for chromosomes D and E had a trivalent frequency of about 30% and pollen stainability of over 70%.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line "TAI-27"revealed by fluorescence in situ hybridization (FISH)

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