Fluorescent in situ hybridization as an aid to introducing alien genetic variation into wheat

Summary Fluorescent in situ hybridization (FISH) has been used to assess the occurrence and frequency of wheat-alien chromosome pairing in a wheat/Thinopyrum bessarabicum hybrid and in wheat/rye hybrids with different levels of chromosome pairing by examining pollen mother cells at metaphase I of meiosis. The use of FISH to identify the presence and size of alien chromatin in a wheat background is also demonstrated.The value of FISH as an aid to the introgression of alien genetic variation into wheat is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization     

Molecular cytogenetic characterization of wheat-Thinopyrum ponticum translocations bearing blue-grained gene(s) induced by r-ray

Eight γ-irradiation-induced Triticum aestivum – Thinopyrum ponticum translocations conveying the blue aleurone were characterized using molecular cytogenetic approach. The size of alien chromosome segments was estimated by genomic in situ hybridization (GISH). The wheat chromosome segments involved in these translocations were clearly identified by two-color fluorescence in situ hybridization (FISH) with the probes of pAs1 and pSc119.2 (or pHvG38). Most of the detected translocations were reciprocal translocations involving wheat chromosomes 1B, 2D, 3A, 4A, 5B, 6B, 6D and 7A. This series of blue-grained wheat translocation lines would be useful in theoretical studies and wheat chromosome engineering breeding.     

Transfer of rust resistance from Aegilops ovata into bread wheat (Triticum aestivum L.) and molecular characterisation of resistant derivatives

An interspecific cross was made to transfer leaf rust and stripe rust resistance from an accession of Aegilops ovata (UUMM) to susceptible Triticum aestivum (AABBDD) cv. WL711. The F₁was backcrossed to the recurrent wheat parent, and after two to three backcrosses and selfing, rust resistant progenies were selected. The C-banding study in a uniformly leaf rust and stripe rust resistant derivative showed a substitution of the 5M chromosome of Ae. ovata for 5D of wheat. Analysis of rust resistant derivatives with mapped wheat microsatellite makers confirmed the substitution of 5M for 5D. Some of these derivatives also possessed one or more of the three alien translocations involving 1BL, 2AL and 5BS wheat chromosomes which could not be detected through C-banding. A translocation involving 5DSof wheat and the substituted chromosome 5M of Ae. ovata was also observed in one of the derivatives. Susceptibility of this derivative to leaf rust showed that the leaf rust resistance gene(s) is/are located on short arm of 5M chromosome of Ae. ovata. Though the Ae. ovatasegment translocated to 1BL and 2AL did not seem to possess any rust resistance gene, the alien segment translocated to 5BS may also possess gene(s) for rust resistance. The study demonstrated the usefulness of microsatellite markers in characterisation of interspecific derivatives. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detection of wheat-barley translocations by genomic in situ hybridization in derivatives of hybrids multiplied in vitro

Wheat-barley translocations were identified by genomicin situ hybridization (GISH) in backcross progenies originating from in vitro regenerated wheat (Triticum aestivum L. cv. Chinese Spring) × barley (Hordeum vulgare L. cv. Betzes) hybrids. The regenerated hybrids were pollinated with the wheat line Martonvásári 9 kr1. Five translocated wheat-barley chromosomes were recovered among 51 BC₂F₂ progeny from the in vitro regenerated wheat × barley hybrids. All were single breakpoint translocations with the relative positions of the breakpoints ranging from the centromere to about 0.8 of the relative arm length. Of the four translocations with intercalary breakpoints, three were transfers of terminal barley segments to wheat chromosomes; one was a transfer of a terminal wheat segment to a barley chromosome. Because of the absence of diagnostic N-bands, the identity of three barley segments could not be determined; in one translocation the barley chromosome involved had a NOR so it must have been 5H or 6H, and the centric translocation was 4HS.2BL. Following selfing, homozygotes of four translocations were selected. The experiment suggests that in vitro culture conditions are conducive for major genome rearrangements in wheat-barley hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ph I-induced transfer of leaf and stripe rust-resistance genes from Aegilops triuncialis and Ae. geniculata to bread wheat

Leaf and stripe rusts are severe foliar diseases of bread wheat. Recently, chromosomes 5M^g from the related species Aegilops geniculata that confers resistance to both leaf and stripe rust and 5U^t from Ae. triuncialis conferring resistance to leaf rust have been transferred to bread wheat in the form of disomic DS5M^g(5D) and DS5U^t(5A) chromosome substitution lines. The objective of this study was to shorten the alien segments in these lines using Ph ^I-mediated, induced homoeologous recombination. Putativerecombinants were evaluated for their rust resistance, and by genomic in situ hybridization and microsatellite analyses. One agronomically useful wheat-Ae. geniculata recombinant resistant to leaf and stripe rust was identified that had only a small terminal segment of the 5M^gL arm transferred to the long arm of an unidentified wheat chromosome. This germplasm can be used directly in breeding programs. Only one leaf rust-resistant wheat-Ae. triuncialis recombinant, which consists of most of the complete 5U^t chromosome with a small terminal segment derived from 5AS, was identified. This germplasm will need further chromosome engineering before it can be used in wheat improvement. This revised version was published online in August 2006 with corrections to the Cover Date.     

Recent advances in alien gene transfer in wheat

Summary The recent advances in alien gene transfer from distantly-related species into wheat are reviewed in the present paper. The main achievements during the last ten years include the great expansion of the range of wide hybridization and development of new techniques for production and characterization of wheat-alien chromosome translocations. Updated results of wide hybridization since 1983 and comprehensive characterization of wheat-alien translocation lines in our laboratory are compiled. The future outlook for alien gene transfer in wheat is also discussed.     

Fluorescent in situ hybridization — a useful aid to the introduction of alien genetic variation into wheat

Summary Fluorescent in situ hybridization (FISH) of DNA to plant chromosomes has proved to be a powerful cytogenetic tool. The value of fluorescent in situ hybridization of total genomic DNA (GISH) of related species is demonstrated in the determination of wheat/alien chromosome pairing in hybrids. Its use for assessing the relative merits of the various genes that affect chromosome pairing is also shown.The ability of GISH to identify the presence in wheat of whole alien chromosomes or alien chromosome segments is illustrated. The potential of FISH for detecting repeated DNA sequences, low copy sequences and single copy genes is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization     

Development and characterization of a <Emphasis Type="Italic">Triticum aestivum</Emphasis>-<Emphasis Type="Italic">Haynaldia villosa</Emphasis> translocation line T4VS⋅4DL conferring resistance to wheat spindle streak mosaic virus

Previous studies showed that a T. aestivum-H. villosa disomic substitution line DS4V(4D) showed a high level of resistance to wheat spindle streak mosaic virus (WSSMV). By crossing DS4V(4D) with the common wheat variety Yangmai #5, plants were obtained that were double monosomic for chromosomes 4V and 4D. Univalents are prone to misdivision at the centromere, and fusion of the derived telocentric chromosomes leads to the production of Robertsonian whole-arm translocations. We screened the progenies of such double monosomic plants by C-banding and genomic in situ hybridization and identified one compensating translocation where the short arm of 4V was translocated to the long arm of 4D of wheat, T4VS⋅4DL. RFLP analysis using the group-4 specific probe BCD110 was used to confirm the translocation. The T4VS⋅4DL translocation stock, accessioned as NAU413, is highly resistant to WSSMV and is also of good agronomic type. The WSSMV resistance gene located on 4VS was designated Wss1.     

Sequential combinations of C-banding and in situ hybridization and their use in the detection of interspecific introgressions into wheat

Summary The sequential combination of C-banding and in situ hybridization techniques applied in this or in a reverse order, are used to recognize targeted chromosomal regions in cereals. Both methods are described whereby standard chromosome squash preparations are followed by: i) C-banding technique using Leishman stain and a slightly modified in situ hybridization technique using biotin-labeled DNA probes, or ii) fluorescence in situ hybridization technique and C-banding. Both approaches have been successfully used onto mitotic chromosomes of rye and wheat resulting suitable for both their identification and detection of targeted sites.     

Wheat chromosome engineering at the 4x level: the potential of different alien gene transfers into durum wheat

Summary With the aim of making the point on feasibility and relative success of alien transfers into durum wheat via chromosome engineering, three transfer works, differing in origin and content of the alien introduction and in the transfer strategy adopted, are described. For the transfer of a powdery mildew resistance gene, Pm13, originating from Aegilops longissima and previously transferred to common wheat chromosome 3B, as well as for that of the leaf rust resistance gene Lr19 and its associated Yp (yellow pigment) gene, deriving from Ag. elongatum and introduced into 7A, the common wheat recombinants were employed as donors, from which the alien segments were homologously transferred into durum genotypes. On the other hand, for the transfer of common wheat chromosome ID seed storage protein genes, ph1 mediated homoeologous recombination was repeatedly induced. This resulted in loss of individuals, including potentially desirable recombinants, probably due to imbalances created by the ph1 condition. However, recovered Gli-D1/Glu-D3 tetraploid recombinants exhibited normal transmission and fertility. Preliminary evidence indicates a normal behaviour also for Glu-D1 5+10 putative recombinants. Similarly, there was no negative impact from the transfer of the Pm13 gene, which has been successfully pyramided into Pm4a durum varieties. On the contrary, transfer of the Ag. elongatum segment showed normal female but almost no male transmission in one durum genotype. This in spite of the fact that the alien segment, proved to be, through in situ hybridization, considerably longer than previously believed, should contain an Sd-1 gene, causing preferential transmission in common wheat. While its behaviour is being checked in other durum genotypes, shortening of the alien segment, through ph1 induced recombination, is also being carried out. Possible causes of the severe negative selection that this alien transfer seemingly encounters at the tetraploid level are discussed.     

Aneuploid and other cytological tester sets in rye

Summary Cytological tester sets include series of aneuploids (nullisomics, monosomics, trisomics of different types, tetrasomies), series of rearranged chromosomes (translocations, inversions, duplications, deficiencies) and series of chromosomes recognizable by specific microscopically visible markers (C- or other banding, molecular markers). In rye, only a few (mainly tertiary and telocentric) monosomics and no viable nullisomics have been found. Several sets of primary trisomics and some telocentric sets, usually not fully complete, have been developed, but few are still available for gene localization. A few tertiary trisomics have been derived from translocation heterozygotes. Extensively used are different sets of additions of rye chromosomes to wheat. A relatively widely distributed set of marked chromosomes is the Wageningen translocation tester set, complemented with translocations from different other institutions. A disadvantage of rye translocations is insufficient heterozygote semisterility. Series of otherwise rearranged chromosomes have not been reported. Sets of lines with chromosomes conspicuously differing from the standard C-banding pattern have been obtained. Molecular markers are available for most rye chromosome, but lack of heterozygosity, necessary for classification after in situ hybridization is a restriction for use as cytological testers. In the cases of most translocations, C-banding and in situ molecular markers, each separate plant in a segregating population must be screened cytologically, whereas with aneuploid markers or with translocations having sufficient heterozygote semisterility, analyzing segregations is sufficient.     

The derivation of compensating translocations involving homoeologous group 3 chromosomes of wheat and rye

Summary The Sr27 translocation in WRT238 was found to consist of chromosome arms 3RS of rye and 3AS of common wheat. An attempt was made to purposely produce compensating translocations having 3RS and a wheat homoeologous group 3L arm. To achieve this, plants, double monosomic for 3R and a wheat homoeologous group 3 chromosome, were irradiated (7.5 Gy gamma rays) or left untreated before being used to pollinate stem rust susceptible testers. Segregation for stem rust resistance was studied to identify F₂ families with Sr27-carrying translocated chromosomes, these were confirmed by means of C-banding. Compensating translocations 3RS3AL and 3RS3BL) were obtained readily and at similar frequencies from untreated and irradiated plants (respectively, 7.2% and 9.3%). Both translocation types have impaired transmission and segregate approximately 3: 2 (present: absent) in the F₂.     

Aneuploid and other cytological tester sets in rye

Summary Cytological tester sets include series of aneuploids (nullisomics, monosomics, trisomics of different types, tetrasomics), series of rearranged chromosomes (translocations, inversions, duplications, deficiencies) and series of chromosomes recognizable by specific microscopically visible markers (C-or other banding, molecular markers). In rye, only a few (mainly tertiary and telocentric) monosomics and no viable nullisomics have been found. Several sets of primary trisomics and some telocentric sets, usually not fully complete, have been developed, but few are still available for gene localization. A few tertiary trisomics have been derived from translocation heterozygotes. Extensively used are different sets of additions of rye chromosomes to wheat. A relatively widely distributed set of marked chromosomes is the Wageningen translocation tester set, complemented with translocations from different other institutions. A disadvantage of rye translocations is insufficient heterozygote semisterility. Series of otherwise rearranged chromosomes have not been reported. Sets of lines with chromosomes conspicuously differing from the standard C-banding pattern have been obtained. Molecular markers are available for most rye chromosome, but lack of heterozygosity, necessary for classification afterin situ hybridization is a restriction for use as cytological testers. In the cases of most translocations, C-banding andin situ molecular markers, each separate plant in a segregating population must be screened cytologically, whereas with aneuploid markers or with translocations having sufficient heterozygote semisterility, analyzing segregations is sufficient.     

Metaphase-I analysis of a Triticum aestivum x T. monococcum hybrid by the C-banding technique

Summary The meiotic pairing behaviour at metaphase I of a Triticum aestivum×Triticum monococcum hybrid has been studied by means of the C-banding technique to ascertain the homology between the chromosomes in the A genome of the two species. The technique allowed the A and B genome chromosomes and the 2D, 3D and 5D chromosomes to be identified. Differences in the level of chromosome pairing in the A genome were noted. The T. monococcum 4A chromosome did not pair with any of the T. aestivum chromosomes in any of the metaphase I cells analysed. Two reciprocal translocations between the 2B and 2D chromosomes on one side and the 2A and 3D on the other side have been identified. The usefulness of the C-banding technique in the study of chromosome homology among species related to wheat is discussed.     

Identifying the alien chromosomes in wheat – Leymus multicaulis derivatives using GISH and RFLP techniques

Genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) were used to identify the Leymus multicaulis (XXNN, 2n = 28) chromosomes in wheat-L. muliticaulis derivatives. Fifteen lines containing L. multicaulis alien chromosomes or chromosomal fragments were identified. All alien chromosomes or fragments in these 15 lines were from the X genome and none were from the N genome. Eleven L. multicaulis disomic addition lines and four translocation-addition lines were identified with chromosome rearrangements among homoeologous groups 2, 3, 6 and 7. Only homoeologous group 1 lacked rearrangements in addition or translocation chromosomes. The results revealed that translocation in non-homoeologous chromosomes widely exists in the Triticeae and therefore it is necessary to identify the alien chromosomes (segments) in a wheat background using these combined techniques. During the course of the work, probe PSR112, was found to detect X genome addition lines involving L. multicaulischromosomes. This may prove to be a valuable probe for the identification of alien chromosomes in a wheat background. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification for H. villosa chromatin in wheat lines using genomic in situ hybridization, C-banding and gliadin electrophoresis techniques

Detection of H. villosa chromosomes in telosomic addition and translocation lines of common wheat was undertaken using genomic in situ hybridization (GISH), C-banding techniques and polyacrylamide gels electrophoresis. The result of GISH on mitotic metaphase cells of the addition line `95039' indicated that the added telochromosomes originated from H. villosa, and it was probably 6VS or 7Vs of H. villosa according to the C-banding pattern. Furthermore, the analysis of gliadin profiles demonstrated that the telochromosome was 6VS. A pair of 1RS/1BL translocation chromosome was also found in `95039'. In addition, mitotic GISH analysis showed that the 6VS/6AL translocation chromosome remained unchanged after being transferred into new wheat background. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spontaneous wheat/rye translocations from female meiotic products of hybrids between octoploid triticale and wheat

Summary Heptaploid hybrids between octoploid triticale and wheat were backcrossed as female parents with wheat to examine the rye chromosome distribution in the resultant progenies using genomic in situ hybridization (GISH). One hundred and one backcross (BC) seeds were examined and whole rye chromosome additions and substitutions, wheat/rye centric and noncentric translocations and rye telocentric chromosomes were detected. Dicentric wheat/rye translocated chromosomes were also observed. Comparisons were made with previous results on the rye chromosome distribution from male gametes of the same cross and differences were found, where in the female derived population a deficit of plants with more than two rye chromosomes was apparent relative to the anther derived population.     

Cytogenetical studies in wheat XVI. Chromosome location of a new gene for resistance to leaf rust in a Japanese wheat-rye translocation line

Summary A leaf rust resistant wheat-rye translocation stock, ST-1, introduced from Japan, comprised distinct morphological types. One type possessed a T1BL·1RS chromosome with genes Lr26, Yr9 and Sr31. A second type carried a new gene, Lr45, located in a large segment of rye chromosome translocated to wheat chromosome 2A. Its structure was identified as T2AS-2RS·2RL. Despite the homoeology of the 2A and 2R chromosomes and the high level of compensation provided by the translocation, Lr45 was not normally inherited and is probably associated with agronomic deficiencies that will prevent its exploitation in agriculture.Contribution No. 94-509-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, USA.     

Molecular cytogenetic characterization of wheat–<Emphasis Type="Italic">Secale africanum</Emphasis> amphiploids and derived introgression lines with stripe rust resistance

Two amphiploids, AF-1(Triticum aestivum L. cv. Anyuepaideng–Secale africanum Stapf.) and BF-1 (T. turgidum ssp. carthlicum–S. africanum), were evaluated by chromosomal banding and in situ hybridization. The individual S. africanum chromosomes were identified in the BF-1 background by sequential C-banding and genomic in situ hybridization (GISH), and were distinguishable from those of S. cereale, because they exhibited less terminal heterochromatin. Fluorescence in situ hybridization (FISH) using the tandem repeat pSc250 as a probe indicated that only 6R^a of S. africanum contained a significant hybrid signal, whereas S. cereale displayed strong hybridization at the telomeres or subtelomeres in all seven pairs of chromosomes. Extensive wheat–S. africanum non-Robertsonian translocations were observed in both AF-1 and BF-1 plants, suggesting a frequent occurrence of chromosomal recombination between wheat and S. africanum. Moreover, introgression lines selected from the progeny of wheat/AF-1 crosses were resistant when field tested with widely virulent strains of Puccinia striiformis f. sp. tritici. Three highly resistant lines were selected. GISH and C-banding revealed that resistant line L9-15 carried a pair of 1BL.1RS translocated chromosomes. This new type of S. africanum derived wheat–Secale translocation line with resistance to Yr9-virulent strains will broaden the genetic diversity of 1BL.1RS for wheat breeding.     

Non-homoeologous wheat-rye chromosomal translocations conferring resistance to greenbug

Summary C-banding andin situ hybridization were used to determine the chromosomal constitution of the greenbug-resistant germplasm GRS 1204. The results showed that this line had the radiation-induced non-homoeologous wheat-rye translocation chromosomes T2AS-1RS·1RL and T2AL·2AS-1RS. C-banding analysis further revealed the presence of a wheat-Agropyron elongatum translocation chromosome T1BL·1BS-3Ae#1L in line GRS 1204, that was derived from Teewon. The greenbug resistance of line GRS 1204 is similar to that of line GRS 1201 that was earlier shown to have the greenbug resistance geneGb6 located on the 1RS arm of the wheat-rye translocation chromosome T1AL·1RS. BecauseGb6 in line GRS 1204 is present on one of the non-homoeologous translocation chromosomes, agronomically line GRS 1201 should be the better adapted source ofGb6 resistance and be used in cultivar improvement.