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.     

Genetic induction of chromosomal rearrangements in barley chromosome 7H added to common wheat

Chromosome 2C of Aegilops cylindrica induces chromosomal rearrangements in alien chromosome addition lines, as well as in euploid lines, of common wheat. To induce chromosomal rearrangements in barley chromosome 7H, reciprocal crosses were made between a mutation-inducing common wheat line that carries a pair of 7H chromosomes and one 2C chromosome and a 7H disomic addition line of common wheat. Many shrivelled seeds were included in the progeny, which was an indication of the occurrence of chromosome mutations. The chromosomal constitution of the viable progeny was examined by FISH (fluorescence in situ hybridization) using the barley subterminal repeat HvT01 as a probe. Structural changes of chromosome 7H were found in about 15% of the progeny of the reciprocal crosses. The aberrant 7H chromosomes were characterized by a combination of N-banding, FISH and genomic in situ hybridization. Mosaicism for aberrant 7H chromosomes was observed in seven plants. In total, 89 aberrant 7H chromosomes were identified in 82 plants, seven of which had double aberrations. More than half of the plants carried a simple deletion: four short-arm telosomes, one long-arm telosome, and 45 terminal deletions (23 in the short arm, 21 in the long arm, and one involving both arms). About 40% of the aberrations represented translocations between 7H and wheat chromosomes. Twenty of the translocations had wheat centromeres, 12 the 7H centromere, with translocation points in the 7HS (five) and in the 7HL (seven), and the remaining four were of Robertsonian type, three involving 7HS and one with 7HL. In addition, one translocation had a barley segment in an intercalary position of a wheat chromosome, and two were dicentric. The breakpoints of these aberrations were distributed along the entire length of chromosome 7H.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.     

Spontaneous chromosomal rearrangements in cultivated and wild barleys

Summary Four of 1,240 cultivated barley lines collected from different regions of the world and 3 of 120 lines of wild barley, Hordeum spontaneum C. Koch, carry spontaneous reciprocal translocations. Break-point positions and rearrangements in the interchanged chromosomes have been examined by both test crosses and Giemsa banding techniques. The four translocation lines in cultivated barley were all of Ethiopian origin and have the same translocation involving chromosomes 2 and 4. The breakpoints are at the centromeres of both chromosomes, resulting in interchanged chromosomes 2S+4S and 2L+4L (S=short arm, L=long arm). A wild barley line, Spont.II, also has translocated chromosomes 2 and 4 which are broken at the centromeres. The resultant chromosomes are, however, 2S+4L and 2L+4S. Another wild barley line, Spont.S-4, has interchanged chromosomes with breakpoints in the short arm of chromosome 3 and the long arm of chromosome 7. In addition, this line has a paracentric inversion in the short arm of chromosome 7 that includes a part of nucleolar constriction, resulting in two tandemly arranged nucleolar constrictions. The third wild barley line, Spont.S-7, has interchanged chromosomes with breakpoints in the long arms of both chromosomes 3 and 6. The translocated chromosome 3 is metacentric and the translocated chromosome 6 has a long arm similar in length to the long arm of chromosome 7.     

Deletion-based physical mapping of barley chromosome 7H

Chromosomal mutations in barley (Hordeum vulgare, 2n=2x=14, HH) chromosome 7H added to the common wheat (Triticum aestivum, 2n=6x=42, AABBDD) cultivar Chinese Spring were induced genetically by the gametocidal activity of certain alien chromosomes derived from wild species of the genus Aegilops. The rearranged barley chromosomes were characterized by C-banding, FISH and GISH. Twenty two deletion or translocation chromosomes in a hemizygous condition were selected for deletion mapping of 17 AFLP and 28 STS markers that are specific to 7H. Of the 22 breakpoints in chromosome 7H, seven involved the short arm (7HS), 12 the long arm (7HL) and three were in the centromeric region. The seven 7HS breakpoints separated all four 7HS-specific AFLP markers and split the 21 STS markers into six groups. One breakpoint occurred between two STS markers formerly occupying the same position in the genetic map. All seven 7HS breakpoints were separated from each other by either the AFLP or STS markers. The 12 breakpoints in 7HL divided the 13 7HL-specific AFLP markers into seven groups, and the seven STS markers into three groups. On the other hand, the 12 breakpoints in 7HL were divided into six groups by the AFLP markers and into two groups by the STS markers. This deletion-based map was in accordance with previously published genetic and physical maps using the same STS markers. The breakpoints, AFLP markers and STS markers were arrayed in a consistent order. Received: 5 February 2001 / Accepted: 19 February 2001     

中国大麦矮秆种质资源的基因分析Ⅱ．矮秆基因的染色体定位

根据连锁遗传原理,利用全套染色体形态性状状标记系,对２０份中国大麦筹秆南资源的矮秆基因,进行了染色体定位,结果表明,１５份单基因矮杆中,有１份其矮秆基因与宽护颖基因w连锁,位于２（２Ｈ）染色体短臂上;１０份的矮秆基因与uz基因等等位,由３（３Ｈ）长臂携带;４份的矮秆基因与钩芒K ,锁位于４（４Ｈ）长臂上,５份双基因矮秆中,有３份的筹秆基因分别位于２（２Ｈ）短臂和４（４Ｈ）长 臂上;１份的筹秆基因各由其３（３Ｈ）和４（４Ｈ）长臂携带;其余１份的两对矮秆基因,１对与uz基因等位,由于３（３Ｈ）长臂携带,另１对则与宽护颖基因w连锁,位于２（２Ｈ）短臂之上。     

中国大麦矮秆种质资源的基因分析——Ⅱ.矮秆基因的染色体定位

根据连锁遗传原理,利用全套染色体形态性状标记系,对２０份中国大麦矮秆种质资源的矮秆基因,进行了染色体定位。结果表明：１５份单基因矮秆中,有１份其矮秆基因与宽护颖基因Ｚ连锁,位于２（２Ｈ）染色体短臂上;１０份的矮秆基因与ｕｚ基因等位,由３（３Ｈ）长臂携带;４份的矮秆基因与钩芒基因Ｋ连锁,位于４（４Ｈ）长臂上。５份双基因矮秆中,有３份的矮秆基因分别位于２（２Ｈ）短臂和４（４Ｈ）长臂上;１份的矮秆基因各由其３（３Ｈ）和４（４Ｈ）长臂携带;其余１份的两对矮秆基因,１对与ｕｚ基因等位,由３（３Ｈ）长臂携带,另１对则与宽护颖基因ｗ连锁,位于２（２Ｈ）短臂之上。     

Genetic Map of Diploid Wheat, Triticum Monococcum L., and Its Comparison with Maps of Hordeum Vulgare L

A genetic map of diploid wheat, Triticum monococcum L., involving 335 markers, including RFLP DNA markers, isozymes, seed storage proteins, rRNA, and morphological loci, is reported. T. monococcum and barley linkage groups are remarkably conserved. They differ by a reciprocal translocation involving the long arms of chromosomes 4 and 5, and paracentric inversions in the long arm of chromosomes 1 and 4; the latter is in a segment of chromosome arm 4L translocated to 5L in T. monococcum. The order of the markers in the inverted segments in the T. monococcum genome is the same as in the B and D genomes of T. aestivum L. The T. monococcum map differs from the barley maps in the distribution of recombination within chromosomes. The major 5S rRNA loci were mapped on the short arms of T. monococcum chromosomes 1 and 5 and the long arms of barley chromosomes 2 and 3. Since these chromosome arms are colinear, the major 5S rRNA loci must be subjected to positional changes in the evolving Triticeae genome that do not perturb chromosome colinearity. The positional changes of the major 5S rRNA loci in Triticeae genomes are analogous to those of the 18S-5.8S-26S rRNA loci.     

Characterization of a new 4BS.7HL wheat-barley translocation line using GISH, FISH, and SSR markers and its effect on the β-glucan content of wheat

A spontaneous interspecific Robertsonian translocation was revealed by genomic in situ hybridization (GISH) in the progenies of a monosomic 7H addition line originating from a new wheat 'Asakaze komugi' × barley 'Manas' hybrid. Fluorescence in situ hybridization (FISH) with repetitive DNA sequences (Afa family, pSc119.2, and pTa71) allowed identification of all wheat chromosomes, including wheat chromosome arm 4BS involved in the translocation. FISH using barley telomere- and centromere-specific repetitive DNA probes (HvT01 and (AGGGAG)(n)) confirmed that one of the arms of barley chromosome 7H was involved in the translocation. Simple sequence repeat (SSR) markers specific to the long (L) and short (S) arms of barley chromosome 7H identified the translocated chromosome segment as 7HL. Further analysis of the translocation chromosome clarified the physical position of genetically mapped SSRs within 7H, with a special focus on its centromeric region. The presence of the HvCslF6 gene, responsible for (1,3;1,4)-β-D-glucan production, was revealed in the centromeric region of 7HL. An increased (1,3;1,4)-β-D-glucan level was also detected in the translocation line, demonstrating that the HvCslF6 gene is of potential relevance for the manipulation of wheat (1,3;1,4)-β-D-glucan levels.     

Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.     

Molecular and physical mapping of a barley gene on chromosome arm 1 HL that causes sterility in hybrids with wheat.

Addition of the long arm of barley chromosome 1H (1HL) to wheat causes severe meiotic abnormalities and complete sterility of the plants. To map the barley gene responsible for the 1H-induced sterility of wheat, a series of addition lines of translocated 1H chromosomes were developed from the crosses between the wheat 'Shinchunaga' and five reciprocal translocation lines derived from the barley line St.13559. Examination of the seed fertility of the addition lines revealed that the sterility gene is located in the interstitial 25% region of the 1HL arm. The genetic location of the sterility gene was also estimated by physically mapping sequence-tagged site (STS) markers and simple-sequence repeat (SSR) markers with known map locations. The sterility gene is designated Shw (sterility in hybrids with wheat). Comparison of the present physical map of 1HL with two previously published genetic maps revealed a paucity of markers in the proximal 30% region and non-random distribution of SSR markers. Two inconsistencies in marker order were found between the present physical map and the consensus genetic map of group 1 chromosomes of Triticeae. On the basis of the effects on meiosis and chromosomal location, the relationship of the present sterility gene with other fertility-related genes of Triticeae is discussed.     

Linkage map construction involving a reciprocal translocation

This paper is concerned with a novel statistical–genetic approach for the construction of linkage maps in populations obtained from reciprocal translocation heterozygotes of barley (Hordeum vulgare L.). Using standard linkage analysis, translocations usually lead to ‘pseudo-linkage’: the mixing up of markers from the chromosomes involved in the translocation into a single linkage group. Close to the translocation breakpoints recombination is severely suppressed and, as a consequence, ordering markers in those regions is not feasible. The novel strategy presented in this paper is based on (1) disentangling the “pseudo-linkage” using principal coordinate analysis, (2) separating individuals into translocated types and normal types and (3) separating markers into those close to and those more distant from the translocation breakpoints. The methods make use of a consensus map of the species involved. The final product consists of integrated linkage maps of the distal parts of the chromosomes involved in the translocation.     

Cytogenetic diversity of SSR motifs within and between Hordeum species carrying the H genome: H. vulgare L. and H. bulbosum L.

Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)—(AG) _n , (AAG) _n , (ACT) _n and (ATC) _n —in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.     

Multiple reconstruction of barley karyotype resulting in complete cytological marking of the chromosome complement

Summary A new reconstructed barley karyotype, PK88, which is a quadruple homozygote for three unequal translocations, 1–2, 3–4, 5–7, and one pericentric inversion in chromosome 6, was studied. As a result of these chromosome rearrangements, a complete cytological marking of the complement has been achieved. Due to the specific intra or interchromosomal transfer of particular bands, Giemsa staining of somatic chromosomes provided clear-cut indications about the localization of translocation and inversion breakpoints. It was established that the long arms of chromosomes 1, 2, 4, 5 and 7 and the short arm of chromosome 3 have been involved in interchanges 1–2, 3–4, and 5–7. The breakpoints of pericentric inversion proved to be located proximally to the short (satellite) arm and distally in the long arm of chromosome 6. PK-88 offers an essential gain in resolution power and extension of the areas of application in cytogenetics over other reconstructed karyotypes produced so far in barley.     

Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected complexity in a rye translocation used in chromosome mapping

Using fluorescence in situ hybridization (FISH) with probe pScT7, three different 5S rDNA loci were detected in the satellite of rye chromosome 1R (5SDna-R1) and in the short arms of chromosomes 3R (5SDna-R3) and 5R (5SDna-R2) respectively. All three loci showed polymorphism for the hybridization signal intensity. In order to determine the localization of these rye 5S rDNA multigene loci with higher precision within the corresponding chromosome arms, the probe pScT7 was physically mapped by FISH in relation to the following five translocations (Wageningen Tester Set): T850W (1RS/4RL), T248W (1RS/6RS), T273W (1RS/5RL), T305W (2RS/5RS) and T240W (3RS/5RL). Accurate physical maps of the translocation breakpoints had previously been made using electron microscope analysis of spread pachytene synaptonemal complexes of heterozygotes for the different translocations. The results indicate that locus 5SDna-R3 is located between the breakpoint of translocation T240W and the telomere, whereas locus 5SDna-R2 is located between the breakpoint of translocation T305W and the centromere, the hybridization of probe pScT7 on T305W translocated chromosomes demonstrating the complex nature of this translocation. On the other hand, the simultaneous detection of probes pScT7 and pTA71 (18S-5.8S-26S rDNA) with two different fluorochromes, indicated that the breakpoints of translocations T850W and T248W are located between loci Nor-R1 and 5SDna-R1.     

Chromosomal assignment and deletion mapping of barley EST markers

From about 10000 PCR-based EST markers of barley we chose 1421 EST markers that were demonstrated to be amplified differently by PCR between wheat (Triticum aestivum cv. Chinese Spring) and barley (Hordeum vulgare cv. Betzes). We assigned them to the seven barley chromosomes (1H to 7H) by PCR analysis using a set of wheat-barley chromosome addition lines. We successfully assigned 701 (49.3%) EST markers to the barley chromosomes: 75 to 1H, 127 to 2H, 119 to 3H, 94 to 4H, 108 to 5H, 81 to 6H and 97 to 7H. By using a set of Betzes barley telosomic addition lines of Chinese Spring, we could successfully determine the chromosome-arm (S or L) location of at least 90% of the EST markers assigned to each barley chromosome. We conducted a trial mapping using 90 EST markers assigned to 7HS (49) or 7HL (41) and 19 wheat lines carrying 7H structural changes. More EST markers were found in the distal region than in the proximal region.     

Molecular cytogenetic analysis of Aegilops cylindrica host.

The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.     

Localization of translocation breakpoints in somatic metaphase chromosomes of barley

Karyotype analyses based on staining by acetocarmine followed by Giemsa N-banding of somatic metaphase chromosomes of Hordeum vulgare L. were carried out on 61 reciprocal translocations induced by X-irradiation. By means of computer-based karyotype analyses all of the 122 breakpoints could be localized to defined sites or segments distributed over the seven barley chromosomes. The pre-definition of translocations with respect to their rearranged chromosome arms from other studies rendered it possible to define the break positions even in translocations having exchanged segments equal in size and the breakpoints located distally to any Giemsa band or other cytological marker. The breakpoints were found to be non-randomly spaced along the chromosomes and their arms. All breaks but one occurred in interband regions of the chromosomes, and none of the breaks was located directly within a centromere. However, short and long chromosome arms recombined at random. An improved tester set of translocations depicting the known break positions of most distal location is presented.