Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Molecular cytogenetic identification of a novel dwarf wheat line with introgressed <Emphasis Type="Italic">Thinopyrum ponticum</Emphasis> chromatin

Novel dwarfing germplasms and dwarfing genes are valuable for the wheat breeding. A novel semi-dwarf line, 31505-1, with reduced height compared with its common wheat parent, was derived from a cross between common wheat and Thinopyrum ponticum. Cytological studies demonstrated that 31505-1 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) analysis showed that 31505-1 had no large Th. ponticum chromosome fragments. Fluorescence in situ hybridization (FISH) results revealed the absence of a pAs1 hybridization band on 2DL chromosome of 31505-1. Two SSR markers (Xwmc41 and Xcfd168) and two STS markers (Xmag4059 and Xmag3596), which were located on 2D chromosome, amplified unique bands of Th. Ponticum in 31505-1. These revealed presence of an introgressed Th. ponticum segment in 2DL chromosome of dwarf line 31505-1, although the alien segment could not be detected by GISH.     

Molecular cytogenetic characterization of two high protein wheat-<Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> partial amphiploids

Fluorescence and genomic in situ hybridization (FISH and GISH) were used to establish the cytogenetic constitution of two wheat × Thinopyrum intermedium partial amphiploids H95 and 55(1-57). Both partial amphiploids are high-protein lines having resistance to leaf rust, yellow rust and powdery mildew and have in total 56 chromosomes per cell. Repetitive DNA probes (pTa71, Afa family and pSc119.2) were used to identify the individual wheat chromosomes and to reveal the distribution of these probes within the alien chromosomes. FISH detected 6B tetrasomy in H95 and a null (1D)-tetrasomy (1B) in 55(1-57). GISH was carried out using biotin labeled Th. intermedium DNA and digoxigenin labeled Pseudoroegneria spicata DNA as probes, subsequently. GISH results revealed 44 wheat chromosomes and four Thinopyrum chromosome pairs, including three S and one J chromosome pairs in line H95. Line 55(1-57), contained 42 wheat chromosomes and six Th. intermedium pairs, including two S and one J^S pairs. Additionally, two identical translocated chromosome pairs with diminished affinity to the alien chromatin were detected in both amphiploids. Another two translocations were found in 55(1-57), with satellite sections from the Thinopyrum J genome.     

The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers.

Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Use of fluorescence in situ hybridization as a tool for introgression analysis and chromosome identification in coffee (Coffea arabica L.).

Fluorescence in situ hybridization (FISH) was used to study the presence of alien chromatin in interspecific hybrids and one introgressed line (S.288) derived from crosses between the cultivated species Coffea arabica and the diploid relatives C. canephora and C. liberica. In situ hybridization using genomic DNA from C. canephora and C. arabica as probes showed elevated cross hybridization along the hybrid genome, confirming the weak differentiation between parental genomes. According to our genomic in situ hybridization (GISH) data, the observed genomic resemblance between the modern C. canephora genome (C) and the C. canephora-derived subgenome of C. arabica (Ca) appears rather considerable. Poor discrimination between C and Ca chromosomes supports the idea of low structural modifications of both genomes since the C. arabica speciation, at least in the frequency and distribution of repetitive sequences. GISH was also used to identify alien chromatin segments on chromosome spreads of a C. liberica-introgressed line of C. arabica. Further, use of GISH together with BAC-FISH analysis gave us additional valuable information about the physical localization of the C. liberica fragments carrying the SH3 factor involved in resistance to the coffee leaf rust. Overall, our results illustrate that FISH analysis is a complementary tool for molecular cytogenetic studies in coffee, providing rapid localization of either specific chromosomes or alien chromatin in introgressed genotypes derived from diploid species displaying substantial genomic differentiation from C. arabica.     

Identification of new T1BL.1RS translocation lines derived from wheat (Triticum aestivum L. cultivar “Xiaoyan No. 6”) and rye hybridization

Two new T1BL.1RS translocation lines, 48112 and 89121, derived from cross between common wheat (Triticum aestivum L.) cultivar “Xiaoyan No. 6” and rye (Secale cereale L.) cultivar “German White”, were developed and identified by using of molecular markers and cytogenetical methods, GISH and FISH. PCR results of primers NOR-R1 specific for rye and Glu-B3 for 1BS detected the presence of 1RS chromatin and absence of 1BS, and primer for gene 1Bx14 in 1BL indicated the existence of chromosome arm 1BL in the two lines. GISH and FISH methods confirmed the replacement of chromosome arm 1BS with 1RS. Further stripe rust resistant test and quality analysis demonstrated that the new 1BL.1RS translocation lines were higher resistant to mixed races of P. striiformis Westend and observed considerable better quality than other popularized T1BL.1RS cultivars in China. The two lines have been used in wheat breeding for high-yield potential and rust resistance.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

Analysis of remote asymmetric somatic hybrids between common wheat and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Callus-derived protoplasts of common wheat (Triticum aestivum L. cv. Hesheng 3) irradiated with ultraviolet light were fused by using the PEG method with cell suspension-derived protoplasts of Arabidopsis thaliana. Regenerated calli and green plants resembling that of wheat were obtained. The hybrid nature of putative calli and plants were confirmed by isozyme, random amplified polymorphic DNA and genomic in situ hybridization (GISH) analyses. GISH results indicated that 1∼3 small chromosome fragments of A. thaliana were found introgression into the terminals of wheat chromosomes, forming highly asymmetric hybrids. Cytoplasmic genome tests did not show any cytoplasmic genetic materials from A. thaliana. However, variations from the normal wheat cytoplasmic genome were found, indicating recombination or rearrangement occurred during the process of somatic hybridization. The chromosome elimination in the asymmetric somatic hybridization of remote phylogenetic relationship was discussed. A miniature inverted-repeat transposable element related sequence was found by chance in the hybrids which might accompany and impact the process of somatic hybridization. Jingyao Deng and Haifeng Cui provided same contribution to this work.     

Introgression of A- and B-genome of tetraploid triticale chromatin into tetraploid rye

An improvement of rye is one of the mainstream goals of current breeding. Our study is concerned with the introduction of the tetraploid triticale (ABRR) into the 4x rye (RRRR) using classical methods of distant crossing. One hundred fifty BC₁F₉ hybrid plants [(4x rye?×?4x triticales)?×?4x rye] obtained from a backcrossing program were studied. The major aim of this work was to verify the presence of an introgressed A- and B- genome chromatin of triticale in a collection of the 4x rye-tiritcale hybrids and to determine their chromosome compositions. In the present study, karyotypes of the previously reported BC₁F₂s and BC₁F₃s were compared with that of the BC₁F₉ generation as obtained after several subsequent open pollinations. The genomic in situ hybridisation (GISH) allowed us to identify 133 introgression forms in which chromosome numbers ranged between 26 and 32. Using four DNA probes (5S rDNA, 25S rDNA, pSc119.2 and pAs1), the fluorescence in situ hybridisation (FISH) was carried out to facilitate an exact chromosome identification in the hybrid plants. The combination of the multi-colour GISH with the repetitive DNA FISH singled out five types of translocated chromosomes: 2A.2R, 4A.4R, 5A.5R, 5B.5R and 7A.7R among the examined BC₁F₉s. The reported translocation lines could serve as valuable sources of wheat chromatin suitable for further improvements of rye.     

Characterization of a leaf rust-resistant wheat–Thinopyrum ponticum partial amphiploid BE-1, using sequential multicolor GISH and FISH

In situ hybridization (multicolor GISH and FISH) was used to characterize the genomic composition of the wheat–Thinopyrum ponticum partial amphiploid BE-1. The amphiploid is a high-protein line having resistance to leaf rust (Puccinia recondita f. sp. tritici) and powdery mildew (Blumeria graminis f. sp. tritici) and has in total 56 chromosomes per cell. Multicolor GISH using J, A and D genomic probes showed 16 chromosomes originating from Thinopyrum ponticum and 14 A genome, 14 B genome and 12 D genome chromosomes. Six of the Th. ponticum chromosomes carried segments different from the J genome in their centromeric regions. It was demonstrated that these alien chromosome segments did not originate from the A, B or D genomes of wheat, so the translocation chromosomes were considered to be J^s type chromosomes carrying segments similar to the S genome near the centromeres. Rearrangements between the A and D genomes of wheat were detected. FISH using Afa family, pSc119.2 and pTa71 probes allowed the identification of all the wheat chromosomes present and the determination of the chromosomes involved in the translocations. The 4A and 7A chromosomes were identified as being involved in intergenomic translocations. The replaced wheat chromosome was identified as 7D. The localization of these repetitive DNA clones on the Th. ponticum chromosomes of the amphiploid was described in the present study. On the basis of their multicolor FISH patterns, the alien chromosomes could be arranged in eight pairs and could also be differentiated unequivocally from each other.     

GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines

Two Brassica napus--Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F₂ progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.     

Transmission and recombination of homeologous<Emphasis Type="Italic"> Solanum sitiens</Emphasis> chromosomes in tomato

The goal of the present experiments was to transfer the chromosomes of Solanum sitiens (syn. Solanum rickii) into cultivated tomato (Lycopersicon esculentum). By crossing an allotetraploid L. esculentum × Solanum sitiens hybrid to sesquidiploid L. esculentum × S. lycopersicoides, a trigenomic hybrid (2n+14=38) was obtained. Analysis of the latter by GISH (genomic in situ hybridization) indicated it contained a full set of 12 S. sitiens chromosomes, plus two extras from S. lycopersicoides. This and other complex hybrids were pollinated with Lycopersicon pennellii-derived bridging lines to overcome unilateral incompatibility. A total of 40 progeny were recovered by embryo rescue, including diploids and aneuploids (up to 2n+8). In order to determine the origin of chromosomes and the location of introgressed segments, progeny were genotyped with RFLP markers. S. sitiens-specific markers on all chromosomes, except 6 and 11, were detected in the progeny. Several S. sitiens chromosomes were transmitted intact, either through chromosome addition (i.e., trisomics) or substitution (i.e., disomics). Recombination between S. sitiens and L. esculentum was detected on most chromosomes, in both diploid and aneuploid progeny. A monosomic alien addition line for S. sitiens chromosome 8 was identified, and the extra chromosome was stably transmitted to approximately 13% of the backcross progeny. This study demonstrates the feasibility of gene transfer from S. sitiens to L. esculentum through chromosome addition, substitution, and recombination in the progeny of complex aneuploid hybrids.Communicated by J.S. Heslop-Harrison     

Molecular cytogenetic characterization of Roegneria ciliaris chromosome additions in common wheat

The development of alien addition lines is important both for transferring useful genes from related species into common wheat and for studying the relationship between alien chromosomes and those of wheat. Roegneria ciliaris (2n=4x=28, S^cS^cY^cY^c) is reported to be a potential source of resistance to wheat scab, which may be useful in wheat improvement. The amphiploid common wheat-R. ciliaris and BC₁F₇ or BC₂F₆ derivatives were screened by C-banding, genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP) for the presence of R. ciliaris chromatin introgressed into wheat. Six lines were identified as disomic chromosome additions (DA), one as a ditelosomic addition (Dt), two as double disomic additions (dDA) and one as a monosomic chromosome addition (MA). RFLP analysis using wheat homoeologous group-specific clones indicated that the R. ciliaris chromosomes involved in these lines belong to groups 1, 2, 3, 5 and 7. The genomic affinities of the added R. ciliaris chromosomes were determined by FISH analysis using the repetitive sequence pCbTaq4.14 as a probe. These data suggest that the R. ciliaris chromosomes in five lines belong to the S^c genome. Based on the molecular cytogenetic data, the lines are designated as DA2S^c#1, Dt2S^c#1L, DA3S^c#1, dDA1S^c#2+5Y^c#1, DA5Y^c#1, DA7S^c#1, DA7Y^c#1 and MA?Y^c#1. Based on the present and previous work, 8 of the 14 chromosomes of R. ciliaris have been transferred into wheat.     

Analysis of nucleolar activity in Agropyron elongatum,its amphiploid with Triticum aestivum and the chromosome addition lines

Summary The nucleolar organizer activity of the Agropyron elongatum, its amphiploid with hexaploid wheat (Triticum aestivum) and the chromosome addition lines is analyzed by the silver-staining procedure. Four Ag-NORs are observed in A. elongatum corresponding to the chromosomes 6E and 7E. In the amphiploid T. aestivum — A. elongatum, eight Ag-NORs are observed which corresponds the wheat chromosomes 1B and 6B and to the elongatum chromosomes 6E and 7E. Thus, there is codominance in the nucleolar organizer activity of the chromosomes of the two species. However, a partial amphiplasty is detected since less than 8 Ag-NORs (7 up to 4) are observed in some metaphase cells; the chromosomes 6E and 7E are occasionally suppressed by wheat chromosomes. This conclusion is confirmed by the behaviour of the addition lines since only in those corresponding to the chromosomes 6E and 7E are the elongatum chromosomes nucleolar active although occasionally they can be suppressed by wheat chromosomes.     

Identification of parental genomes and genomic organization in <Emphasis Type="Italic">Aster microcephalus</Emphasis> var. <Emphasis Type="Italic">ovatus</Emphasis>

The karyotype of diploid Aster iinumae is morphologically similar to that of diploid Aster ageratoides var. ageratoides, however, its chromosome size is apparently smaller (S-type chromosomes versus L-type chromosomes, respectively). The hybrid origin of tetraploid Aster microcephalus var. ovatus (LS-type chromosomes) has previously been suggested by cytogenetics and chloroplast DNA (cp DNA) data. The cp DNA phylogeny also implies that the S-type chromosome is apomorphic, which means that genome size reduction occurred on the evolutionary way to A. iinumae. In this study, we have demonstrated that the chromosome size difference does not depend on the intensity of chromosome condensation but on the DNA content. The simultaneous genomic in situ hybridization (GISH) results show the similarity between S-type chromosomes of A. iinumae and A. microcephalus var. ovatus, and between L-type chromosomes of A. ageratoides and A. microcephalus var. ovatus, which provide additional evidence for A. microcephalus var. ovatus being a tetraploid amphidiploid produced by hybridization between S-type chromosomes and L-type chromosomes. The distribution patterns of Ty1-copia-like retrotransposons were similar in L- and S-type chromosomes. The copies of this retrotransposon dispersed uniformly on all chromosomes, and it is not yet apparent how the Ty1-copia-like retrotransposon affects the size difference between them.     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

用顺序GISH-FISH 技术鉴定小麦-中间偃麦草小片段易位系

利用顺序基因组-重复序列原位杂交技术对1个来自中3不育系和普通小麦恢75杂种后代稳定株系H96276-2的染色体组成进行了分析。以中间偃麦草（Agropyronintermedium）基因组DNA为探针的荧光原位杂交结果表明,H96276-2的体细胞中有42条染色体,包括20对小麦染色体和1对小麦-中间偃麦草易位染色体,中间偃麦草染色体的易位片段位于1对小麦染色体的端部。进而用重复序列探针pSc119进行第2次荧光原位杂交,证明H96276-2中的中间偃麦草染色体易位片段位于小麦2B染色体的短臂上。     

GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus

New Brassica napus inbred lines with different petal colors and with canola quality and increased levels of oleic (∼70%, 10% higher than that of B. napus parent) and linoleic (28%) acids have been developed in the progenies of one B. napus cv. Oro × Orychophragmus violaceus F₅ hybrid plant (2n=31). Their genetic constituents were analyzed by using the methods of genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). No intact chromosomes of O. violaceus origin were detected by GISH in their somatic cells of ovaries and root tips (2n=38) and pollen mother cells (PMCs) with normal chromosome pairing (19 bivalents) and segregation (19:19), though signals of variable sizes and intensities were located mainly at terminal and centromeric parts of some mitotic chromosomes and meiotic bivalents at diakinesis or chromosomes in anaphase I groups and one large patch of chromatin was intensively labeled and separated spatially in some telophase I nuclei and metaphase II PMCs. AFLP analysis revealed that substantial genomic changes have occurred in these lines and O. violaceus–specific bands, deleted bands in ‘Oro’ and novel bands for two parents were detected. The possible mechanisms for these results were discussed.     

Development of <Emphasis Type="Italic">Triticum aestivum-Leymus racemosus</Emphasis> translocation lines using gametocidal chromosomes

Specific chromosomes of certain Aegilops species introduced into wheat genome background may often facilitate chromosome breakage and refusion, and finally result in a variety of chromosome restructuring. Such a phenomenon is commonly called gametocidal effect of the chromosomes. The chromosome 2C of Ae. cylindrica is one of such chromosomes. In the present study, scab resistant wheat-L. racemosus addition lines involving chromosomes Lr.2 and Lr.7 were crossed to wheat-Ae. cylindrica disomic addition line Add2C. Then F₁ hybrids were subsequently backcrossed with wheat cv “Chinese Spring”. BC₁ plants with chromosome structural aberration were identified by C-banding. In the self-pollinated progenies of these plants, three translocation lines were developed and characterized by mitotic and meiotic analysis combined with C-banding and fluorescent in situ hybridization (FISH) using biotin-labeled genomic DNA of L. racemosus as probe. Some other putative translocation lines to be further characterized were also found. The practicability and efficiency of the translocation between wheat and alien chromosomes induced by gametocidal chromosomes, as well as the potential use of the developed alien translocation lines were also discussed.