The genomic composition of Tricepiro, a synthetic forage crop.

Chromosome in situ hybridization (FISH and GISH) is a powerful tool for determining the chromosomal location of specific sequences and for analysing genome organization and evolution. Tricepiro (2n = 6x = 42) is a synthetic cereal obtained by G. Covas in Argentina (1972), which crosses hexaploid triticale (2n = 6x = 42) and octoploid Trigopiro (2n = 8x = 56). Several years of breeding produced a forage crop with valuable characteristics from Secale, Triticum, and Thinopyrum. The aim of this work is to analyse the real genomic constitution of this important synthetic crop. In situ hybridization using total DNA of Secale, Triticum, and Thinopyrum as a probe (GISH) labelled with biotin and (or) digoxigenin showed that tricepiro is composed of 14 rye chromosomes and 28 wheat chromosomes. Small zones of introgression of Thinopyrum on wheat chromosomes were detected. The FISH using the rye repetitive DNA probe pSc 119.2 labelled with biotin let us characterize the seven pairs of rye chromosomes. Moreover, several wheat chromosomes belonging to A and B genomes were distinguished. Therefore, tricepiro is a synthetic hexaploid (2n = 6x = 42) being AABBRR in its genomic composition, with zones of introgression of Thinopyrum in the A genome of wheat.     

Structure of the rye midget chromosome analyzed by FISH and C-banding.

The diminutive "midget" chromosome derived from rye (Secale cereale) was analyzed by C-banding and fluorescence in situ hybridization (FISH) using DNA probe pSau3A9 that is located in the centromeres of cereal chromosomes. FISH signals were detected at one end and overlapped one of the two telomeres of the midget, indicating that the midget is a telocentric chromosome. The FISH and C-banding results show that the centromere of the midget chromosome is smaller than those of normal wheat and rye chromosomes. These results indicate that one of the breakpoints occurred in the middle of the centromere of rye chromosome 1R during generation of the midget.     

Variations of tandem repeat, regulatory element, and promoter regions revealed by wheat-rye amphiploids.

To better understand the evolution of allopolyploids, 4 different combinations between wheat (Triticum aestivum L.) and rye (Secale cereale L.) including 12 F1 hybrids and 12 derived amphiploids were analyzed and compared with their direct parental plants by PCR analysis using 150 wheat SSR (single sequence repeat) markers and by FISH analysis using a rye-specific repetitive sequence (pSc200) as a probe. Nine SSR markers amplified rye-specific fragments whose sizes ranged from 471 bp to 1089 bp. These fragments contain regulatory elements and (or) promoters. Some of these fragments were amplified from all 24 progenies, while others were amplified from a subset of the progenies. The disappearance of rye-specific fragments from some progenies was caused by sequence elimination or DNA modification. Marker Xgwm320 amplified a new fragment (403 bp), a rye-specific tandem repeat, from some of the progenies. Twenty-eight SSR markers displayed microsatellite variation in progenies derived from 'Chinese Spring' x 'Jinzhou-heimai', but none of the 150 SSR markers displayed microsatellite variation in the progenies derived from the other three combinations. FISH signals of pSc200 were eliminated from one telomere/subtelomere of 4 chromosomes of 'Kustro' during allopolyploidization and expanded in amphiploids derived from 'Chinese Spring' x 'AR106BONE'. Thus, allopolyploidization in wheat-rye can be accompanied by rapid variation of tandem repeats, regulatory elements, and promoter regions. The alterations of repetitive sequence pSc200 indicate coordination between the constituent genomes of the newly formed amphiploids. Different genetic backgrounds of parents appear to affect genome changes during allopolyploidization.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

Isolation and chromosomal localization of new MITE-like sequences from <Emphasis Type="Italic">Secale</Emphasis>

A species-specific DNA sequence (marker) that can detect the presence of Secale cereale chromatin in common wheat background was developed by using wheat microsatellite primer Xgwm614. One rye-specific DNA amplified fragment of 416bp (pSa614₄₁₆) was obtained from Secale africanum and a wheat — S. africanum amphiploid. The primer Xgwm614 also gave rise to specific bands in all Chinese Spring-Imperial rye addition lines 1R to 7R. Sequence analysis revealed that pSa614₄₁₆ was strongly homologous to a miniature inverted transposable element (MITE) stowaway-like element. Results of fluorescence in situ hybridization showed that the signal of pSa614₄₁₆ was distributed along all S. cereale. cv Jingzhou chromosomes, but the signal strengths were unbalanced on the seven rye genome chromosomes. This repetitive element may be useful as a molecular marker for the introgression of rye germplasm into the wheat genome.     

Molecular Cytogenetic Identification of Triticum aestivum-Secale cereale Substitution and Addition Lines

Two substitution lines, designated as 930498 and 930483, and one addition line, designated as 930029, via Fo immature embryo culture of Triticum aestivum x octoploid triticale ( x Triti-cosecale Wittmack) were identified. Fluorescence in situ hybridization (FISH) using total genomic DNA of rye ( Secale cereale L. ) as probe corroborated the existence of rye chromosomes, further confirmed through chromosome paring at meiotic metaphase 1, C-banding and glutenin SDS- PAGE. The results demonstrated that the two substitution lines are ID/IR, and the addition line is also IR addition. Rye chromosomes that are distinct to the red-colored wheat chromosomes appear yellow-green at mitotic metaphase after FISH.     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the Individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aeativum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S.cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattem of Secale vavilovli was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.     

Inter- and intra-genomic transfer of small chromosomal segments in wheat-rye allopolyploids

Newly synthesized wheat-rye allopolyploids, derived from Triticum aestivum Mianyang11 × S. cereale Kustro, were investigated by sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) using rye tandem repeat pSc200 and rye genomic DNA as probes, respectively, over the first, second and third allopolyploid generations. FISH signals of pSc200 could be observed at both telomeres/subtelomeres of all 14 chromosomes of the parental rye. In the first allopolyploid generation, there were ten rye chromosomes bearing FISH signals at both telomeres/subtelomeres and four rye chromosomes bearing FISH signals at only one telomere/subtelomere. However, in the second and the third allopolyploid generations, there were 12 rye chromosomes bearing FISH signals at both telomeres/subtelomeres and 2 rye chromosomes bearing FISH signals at only one telomere/subtelomere. Rye telomeric segments were transferred to the centromeric region of wheat chromosomes in some cells and small segments derived from non-telomeric regions of rye chromosome were transferred to the telomeric region of wheat chromosomes in some other cells. These observations indicated that the rye telomeric/subtelomeric region was unstable in newly synthesized wheat-rye allopolyploids and allopolyploidization was accompanied by rapid inter/intra-genomic exchange. The inter-genomic exchange may have occurred in somatic cells.     

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.     

Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.     

Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

Identification of Alien Chromatin and Ribosomal DNA in Wheat by in Situ Hybridization

In situ hybridization was carried out to somatic cells of hexaploid Triticale “Badger”, lB/IR translocation line “Ning 8026” and IR(ID) substitution line “84056-1-36-1” using biotin-labelled total rye genomic DNA and wheat rDNA as probes, the results were as follows: 1. The probe containing the total genomic DNA from rye hybridized to the entire length of all rye chromosomes, as a result of the formation of a brown precipitate over the sites of hybridization, the rye chromosomes could be distinguished from wheat chromosomes counterstained by Wright’s solution, the distinguishable appearance of the wheat and rye chromosomes resulted in an efficient method of detecting rye chromosome or segments in wheat. 2. When the probe PTA 71 containing wheat ribosomal DNA was used to hybridize to somatic chromosomes of "Badger" and “84056-1-36-1”, six signals in “Badger” and eight in “84056-1-36-1” were observed on lB, 6B, 1R and SD, among which lB and 6B showed large in situ signals corresponding to many copies of the genes. 3. The expression behavior of wheat rDNA was found in interphase cells by in situ hybridization.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.).

A diminutive rye chromosome (midget) in wheat was used as a model system to isolate a highly reiterated centromeric sequence from a rye chromosome. Fluorescence in situ hybridization (FISH) shows this sequence localized within all rye centromeres and no signal was detected on wheat chromosomes. DNA sequencing of the repetitive element has revealed the presence of some catalytic domains and signature motifs typical of retrotransposon genes and has been called the Bilby family, representing a diverged family of retrotransposon-like elements. Extensive DNA database searching revealed some sequence similarity to centromeric retrotransposons from wheat, barley, and centromeric repetitive sequences from rice. Very low levels of signal were observed when Bilby was used as a probe against barley, and no signal was detected with rice DNA during Southern hybridization. The abundance of Bilby in rye indicates that this family may have diverged from other distantly related centromeric retrotransposons or incorporated in the centromere but rapidly evolved in rye during speciation. The isolation of a rye retrotransposon also allowed the analysis of centromeric breakpoints in wheat-rye translocation lines. A quantitative analysis shows that the breakpoint in IDS.1RL and 1DL.1RS and recombinant lines containing proximal rye chromatin have a portion of the rye centromere that may contribute to the normal function of the centromeric region.     

Development and molecular cytogenetic identification of new winter wheat--winter barley ('Martonvásári 9 kr1' - 'Igri') disomic addition lines.

This paper describes a series of winter wheat - winter barley disomic addition lines developed from hybrids between winter wheat line Triticum aestivum L. 'Martonvásári 9 kr1' and the German 2-rowed winter barley cultivar Hordeum vulgare L. 'Igri'. The barley chromosomes in a wheat background were identified from the fluorescent in situ hybridization (FISH) patterns obtained with various combinations of repetitive DNA probes: GAA-HvT01 and pTa71-HvT01. The disomic addition lines 2H, 3H, and 4H and the 1HS isochromosome were identified on the basis of a 2-colour FISH with the DNA probe pairs GAA-pAs1, GAA-HvT01, and pTa71-HvT01. Genomic in situ hybridization was used to confirm the presence of the barley chromosomes in the wheat genome. The identification of the barley chromosomes in the addition lines was further confirmed with simple-sequence repeat markers. The addition lines were also characterized morphologically.     

New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat

Subcloning of a clone of the 120-bp family of rye, pSc119, has produced two extremely useful probes. pSc119.1 assays rye-specific dispersed repetitive sequence families. It is present on all seven rye chromosomes and hybridizes to the entire length of each chromosome, with the exception of some telomeres and the nucleolar organiser region. pSc119.2, in contrast, hybridizes predominantly to the telomeric regions of rye chromosomes, with some interstitial sites. Unlike pSc119.1, it assays similar repetitive sequence families in both wheat and rye chromosomes.     

Origin and molecular structure of a midget chromosome in a common wheat carrying rye cytoplasm

The origin and molecular structure of the midget chromosome that is retained in a common wheat with rye cytoplasm, were studied by using fluorescent in situ hybridization (FISH). FISH with biotinylated rye genomic DNA as a probe clearly showed that the midget chromosome had originated from certain part(s) of rye chromosome(s). The midget chromosome did not possess sequences similar to wheat rDNA nor to a rye telomeric sequence with a 350 bp repeat unit. However, another repetitive sequence (120 bp family) of rye was found to occur at one end of the midget chromosome. The telomeric repeat sequences from Arabidopsis thaliana cross-hybridized to both ends of the midget chromosome as well as to wheat chromosomes. From the results obtained in this and previous studies, it is assumed that the midget chromosome originated from part of a rye chromosome, most likely the centromeric region of chromosome 1R, and that the telomeric sequences were synthesized de novo.by R. Appels     

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.