BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.Communicated by P.B. Moens     

Molecular cytogenetic characterization of Aegilops biuncialis and its use for the identification of 5 derived wheat-Aegilops biuncialis disomic addition lines.

The aim of the experiments was to produce and identify different Triticum aestivum-Aegilops biuncialis disomic addition lines. To facilitate the exact identification of the Ae. biuncialis chromosomes in these Triticum aestivum-Ae. biuncialis disomic additions, it was necessary to analyze the fluorescence in situ hybridization (FISH) pattern of Ae. biuncialis (2n = 4x = 28, U(b)U(b)M(b)M(b)), comparing it with the diploid progenitors (Aegilops umbellulata, 2n = 2x = 14, UU and Aegilops comosa, 2n = 2x = 14, MM). To identify the Ae. biuncialis chromosomes, FISH was carried out using 2 DNA clones (pSc119.2 and pAs1) on Ae. biuncialis and its 2 diploid progenitor species. Differences in the hybridization patterns of all chromosomes were observed among the 4 Ae. umbellulata accessions, the 4 Ae. comosa accessions, and the 3 Ae. biuncialis accessions analyzed. The hybridization pattern of the M genome was more variable than that of the U genome. Five different wheat-Ae. biuncialis addition lines were produced from the wheat-Ae. biuncialis amphiploids produced earlier in Martonvásár. The 2M, 3M, 7M, 3U, and 5U chromosome pairs were identified with FISH using 3 repetitive DNA clones (pSc119.2, pAs1, and pTa71) in the disomic chromosome additions produced. Genomic in situ hybridization (GISH) was used to differentiate the Ae. biuncialis chromosomes from wheat, but no chromosome rearrangements between wheat and Ae. biuncialis were detected in the addition lines.     

Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Salix viminalis L. (2n?=?38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afa-family, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

<Emphasis Type="Italic">Agropyron elongatum</Emphasis> chromatin localization on the wheat chromosomes in an introgression line

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F₅ line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1–3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid II-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-03₁₂₉₆ from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.     

Fluorescence in situ hybridization mapping of Avena sativa L. cv. SunII and its monosomic lines using cloned repetitive DNA sequences.

Fluorescent in situ hybridization (FISH) employing multiple probes was used with mitotic or meiotic chromosome spreads of Avena sativa L. cv. SunII and its monosomic lines to produce physical chromosome maps. The probes used were Avena strigosa pAs120a (which hybridizes exclusively to A-genome chromosomes), Avena murphyi pAm1 (which hybridizes exclusively to C-genome chromosomes), A. strigosa pAs121 (which hybridizes exclusively to A- and D-genome chromosomes), and the wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH employing two-by-two combinations of these probes allowed the unequivocal identification and genome assignation of all chromosomes. Ten pairs were found carrying intergenomic translocations: (i) between the A and C genomes (chromosome pair 5A); (ii) between the C and D genomes (pairs 1C, 2C, 4C, 10C, and 16C); and (iii) between the D and C genomes (pairs 9D, 11D, 13D, and 14D). The existence of a reciprocal intergenomic translocation (10C-14D) is also proposed. Comparing these results with those of other hexaploids, three intergenomic translocations (10C, 9D, and 14D) were found to be unique to A. sativa cv. SunII, supporting the view that 'SunII' is genetically distinct from other hexaploid Avena species and from cultivars of the A. sativa species. FISH mapping using meiotic and mitotic metaphases facilitated the genomic and chromosomal identification of the aneuploid chromosome in each monosomic line. Of the 18 analyzed, only 11 distinct monosomic lines were actually found, corresponding to 5 lines of the A genome, 2 lines of the C genome, and 4 lines of the D genome. The presence or absence of the 10C-14D interchange was also monitored in these lines.     

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.     

小偃6号背景下黑麦遗传物质的荧光原位杂交分析

利用普通小麦(Triticum aestivum L.)“小偃6号”与黑麦(Secale cereale L.)品种“德国白粒”杂交,选育出“小偃6号”类型带有黑麦性状的种质材料。应用总基因组原位杂交(GISH)进行检测,在8份材料中探测到黑麦染色质的存在,其中附加系3个,代换系1个,易位系4个;进一步用荧光绿标记探针pSc119.2及荧光红标记探针pAs1的双色荧光原位杂交(FISH)技术,对其中部分品系的染色体组成进行分析鉴定,结果表明:易位系BC116-1是1RS/1BL小麦/黑麦易位系,BC152-1是涉及一条1B染色体的1RS/1BL易位系, 代换系BC97-2是2R(2D)二体代换系;附加系BC122-3附加了一条6R黑麦染色体,一条6B染色体的长臂缺失。同时,对连续的总基因组原位杂交和双色荧光原位杂交技术在小麦育种中的应用进行了讨论。     

Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Although the centromeres of some plants have been investlgated prevlously, our knowledge of the wheat centromere Is still very llmlted. To understand the structure and functlon of the wheat centromere, we used two centromeric repeats （RCS1 and CCS1-5ab） to obtain some centromere-assoclated bacterial artificial chromosome （BAC） clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat （Triticum boeoticum Boiss.; 2n=2x=14） BAC library. Southern hybridization results indicated that, of the 32 candidates, there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization （FISH） analysis Indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromerlc regions in hexaplold wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or Dgenome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific （or rich） sequences dispersing on chromosome arms in the BAC clone TbBACS. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation In the period before metaphase.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Physical mapping of the blue-grained gene(s) from Thinopyrum ponticum by GISH and FISH in a set of translocation lines with different seed colors in wheat.

The original blue-grained wheat, Blue 58, was a substitution line derived from hybridization between common wheat (Triticum aestivum L., 2n=6x=42, ABD) and tall wheatgrass (Thinopyrum ponticum Liu & Wang=Agropyron elongatum, 2n=10x=70, StStEeEbEx), in which one pair of 4D chromosomes was replaced by a pair of alien 4Ag chromosomes (unknown group 4 chromosome from A. ponticum). Blue aleurone might be a useful cytological marker in chromosome engineering and wheat breeding. Cytogenetic analysis showed that blue aleurone was controlled by chromosome 4Ag. GISH analysis proved that the 4Ag was a recombination chromosome; its centromeric and pericentromeric regions were from an E-genome chromosome, but the distal regions of its two arms were from an St-genome chromosome. On its short arm, there was a major pAs1 hybridization band, which was very close to the centromere. GISH and FISH analysis in a set of translocation lines with different seed colors revealed that the gene(s) controlling the blue pigment was located on the long arm of 4Ag. It was physically mapped to the 0.71-0.80 regions (distance measured from the centromere of 4Ag). The blue color is a consequence of dosage of this small chromosome region derived from the St genome. We speculate that the blue-grained gene(s) could activate the anthocyanin biosynthetic pathway of wheat.     

Alterations and Abnormal Mitosis of Wheat Chromosomes Induced by Wheat-Rye Monosomic Addition Lines

Background

Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported.

Methodology/Principal Findings

Octoploid triticale was derived from common wheat T. aestivum L. ‘Mianyang11’×rye S. cereale L. ‘Kustro’ and some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in ‘Mianyang11’. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line.

Conclusions/Significance

These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat.     

Cytogenetic analysis of Aegilops chromosomes, potentially usable in triticale (X Triticosecale Witt.) breeding

Chromosome identification using fluorescence in situ hybridization (FISH) is widely used in cytogenetic research. It is a diagnostic tool helpful in chromosome identification. It can also be used to characterize alien introgressions, when exercised in a combination with genomic in situ hybridization (GISH). This work aims to find chromosome identification of Aegilops species and Aegilops × Secale amphiploids, which can be used in cereal breeding as a source of favourable agronomic traits. Four diploid and two tetraploid Aegilops species and three Aegilops × Secale hybrids were analysed using FISH with pSc119.2, pAs1, 5S rDNA and 25S rDNA clones to differentiate the U-, M-, S^sh- and D-subgenome chromosomes of Aegilops genus. Additionally, GISH for chromosome categorization was carried out. Differences in the hybridization patterns allowed to identify all U-, M-, S^sh- and D-subgenome chromosomes. Some differences in localization of the rDNA, pSc119.2 and pAs1 sequences between analogue subgenomes in diploid and tetraploid species and Aegilops × Secale hybrids were detected. The hybridization pattern of the M and S genome was more variable than that of the U and D genome. An importance of the cytogenetic markers in plant breeding and their possible role in chromosome structure, function and evolution is discussed.     

Identification of wheat-barley translocations by sequential GISH and two-colour FISH in combination with the use of genetically mapped barley SSR markers.

Five wheat-barley translocations in a wheat background were characterized through the combination of cytogenetic and molecular genetic approaches. The wheat chromosome segments involved in the translocations were identified using sequential GISH and two-colour FISH with the probes pSc119.2 and pAs1. The barley chromatin in these lines was identified using SSR markers. A total of 45 markers distributed over the total barley genome were selected from a recently published linkage map of barley and tested on the translocation lines. The following translocations were identified: 2DS.2DL-1HS, 3HS.3BL, 6BS.6BL-4HL, 4D-5HS, and 7DL.7DS-5HS. Wheat-barley disomic and ditelosomic addition lines for the chromosomes 3HS, 4H, 4HL, 5H, 5HL, and 6HS were used to determine the correct location of 21 markers and the position of the centromere. An intragenomic translocation breakpoint was detected on the short arm of the barley chromosome 5H with the help of SSR marker analysis. Physical mapping of the SSR markers on chromosomes 1H and 5H was carried out using the intragenomic and the interspecific translocation breakpoints, as well as the centromere, as physical landmarks.     

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.     

High-resolution FISH on super-stretched flow-sorted plant chromosomes

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.     

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

 Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae. Received: 21 February 1996 / Accepted: 12 July 1996     

Identification and chromosomal location of tandemly repeated DNA sequences in Allium cepa

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.