A stable dicentric chromosome: Both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric configuration

A stable, dicentric human chromosome, which is known from light microscopy to show a 50:50 distribution between monocentric/dicentric appearance, was examined by conventional electron microscopy and after labelling the centromere with anticentromere antibodies from CREST serum. Both centromeres of the chromosome developed kinetochores whether in monocentric or dicentric configuration. The eight monocentrics observed had all developed kinetochores at the centromere outside the constriction; at least six of them also had kinetochores at the centromere in the constriction. The dicentrics from glutaraldehyde fixed cells had spindle microtubules attached to both kinetochore sets irrespective of monocentric/dicentric configuration. The chromosome thus appeared to use both centromeres, either equally or with one serving a chromatid adhesion function while the second was used for transport along the spindle.     

Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation.

To test the centromere misdivision model of isochromosome formation, we have defined the breakpoints of cytogenetically monocentric and dicentric Xq isochromosomes (i(Xq)) from Turner syndrome probands, using FISH with cosmids and YACs derived from a contig spanning proximal Xp. Seven different pericentromeric breakpoints were identified, with 10 of 11 of the i(Xq)s containing varying amounts of material from Xp. Only one of the eight cytogenetically monocentric i(Xq)s demonstrated a single alpha-satellite (DXZ1) signal, consistent with classical models involving centromere misdivision. The remaining seven were inconsistent with such a model and had breakpoints that spanned proximal Xp11.21: one was between DXZ1 and the most proximal marker, ZXDA; one occurred between the duplicated genes, ZXDA and ZXDB; two were approximately 2 Mb from DXZ1; two were adjacent to ALAS2 located 3.5 Mb from DXZ1; and the largest had a breakpoint just distal to DXS1013E, indicating the inclusion of 8 Mb of Xp DNA between centromeres. The three cytologically dicentric i(Xq)s had breakpoints distal to DXS423E in Xp11.22 and therefore contained > or = 12 Mb of DNA between centromeres. These data demonstrate that the majority of breakpoints resulting in i(Xq) formation are in band Xp11.2 and not in the centromere itself. Therefore, we hypothesize that the predominant mechanism of i(Xq) formation involves sequences in the proximal short arm that are prone to breakage and reunion events between sister chromatids or homologous X chromosomes.     

Behavior of dicentric chromosomes in budding yeast

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.     

Two dicentric Y isochromosomes,one without the Yqh heterochromatic segment

Summary Two women with primary amenorrhoea and few other stigmata of Turner's syndrome were found to be chromosome mosaics: 45,X/46,X,idic(Y). In Case 1, the dicentric isochromosome Y was found to have a long-arm breakpoint of formation. This structure was interpreted as containing two Y short arms and centromeres separated by a region derived from the proximal Y long arm. One of the centromeres in the Case 1 —idic(Y) was suppressed in 80% of cells in blood, and in these cells it appeared as a regular Y-shaped chromosome. In Case 2 the idic(Y) was derived by a short-arm breakpoint of formation. In all the dicentrics of this case with one primary constriction (functional monocentrics) there was a single Cd band. In the 10% of dicentrics with two primary constrictions, there were two Cd bands. It is argued that the instability of sex isochromosomes is due to this functional dicentricity in some cells. These cases are compared with 42 other Y isochromosomes with various short- and long-arm breakpoints of formation. It is suggested that some of the nonheterochromatic, nonfluorescent Y chromosomes previously reported may be explained as dicentric i(Y) with proximal long-arm breakpoints of formation and one suppressed centromere.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications.

We studied 16 cases of 8p duplications, with a karyotype 46,XX or XY,dup(8p), associated with mental retardation, facial dysmorphisms, and brain defects. We demonstrate that these 8p rearrangements can be either dicentric (6 cases) with the second centromere at the tip of the short arm or monocentric (10 cases). The distal 8p23 region, from D8S349 to the telomere, including the defensin 1 locus, is deleted in all the cases. The region spanning from D8S252 to D8S265, at the proximal 8p23 region, is present in single copy, and the remaining part of the abnormal 8 short arm is duplicated in the dicentric cases and partially duplicated in the monocentric ones. The distal edge of the duplication always spans up to D8S552 (8p23.1), while its proximal edge includes the centromere in the dicentric cases and varies from case to case in the monocentric ones. The analysis of DNA polymorphisms indicates that the rearrangement is consistently of maternal origin. In the deleted region, only paternal alleles were present in the patient. In the duplicated region, besides one paternal allele, some loci showed two different maternal alleles, while others, which were duplicated by FISH analysis, showed only one maternal allele. We hypothesize that, at maternal meiosis I, there was abnormal pairing of chromosomes 8 followed by anomalous crossover at the regions delimited by D8S552 and D8S35 and by D8S252 and D8S349, which presumably contain inverted repeated sequences. The resulting dicentric chromosome, 8qter-8p23.1(D8S552)::8p23.1-(D8S35)-8q ter, due to the presence of two centromeres, breaks at anaphase I, generating an inverted duplicated 8p, dicentric if the breakage occurs at the centromere or monocentric if it occurs between centromeres.     

Centromeric inactivation in a dicentric human Y;21 translocation chromosome

A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event. Received: 30 January 1997; in revised form: 3 April 1997 / Accepted: 8 May 1997     

Deletion of specific sequences or modification of centromeric chromatin are responsible for Y chromosome centromere inactivation

Summary Stable dicentric chromosomes behave as monocentrics because one of the centromeres is inactive. The cause of centromere inactivation is unknown; changes in centromere chromatin conformation and loss of centromeric DNA elements have been proposed as possible mechanisms. We studied the phenomenon of inactivation in two Y centromeres, having as a control genetically identical active Y centromeres. The two cases have the following karyotypes: 45,X/46,X,i(Y)(q12) and 46,XY/ 47,XY,+t(X;Y)(p22.3;p11.3). The analysis of the behaviour of the active and inactive Y chromosome centromeres after Da-Dapi staining, CREST immunofluorescence, and in situ hybridization with centromeric probes leads us to conclude that, in the case of the isochromosome, a true deletion of centromeric chromatin is responsible for its stability, whereas in the second case, stability of the dicentric (X;Y) is the result of centromere chromatin modification.     

A tdic(5;15)(p31;p11) chromosome showing variation for constriction in the centromeric regions in a patient with the cri du chat syndrome.

Some dicentric chromosomes show only one primary constriction at metaphase and behave in cell division as if they are monocentric. The few previous reports of tdic (translocation dicentric) chromosomes showing one morphologic indicate that among the cells of an individual the same centromere consistently shows the primary constriction. The present case deals with a tdic(5;15)(p13;p11) chromosome that is an exception to this pattern. Scoring 98 GTG-, C-, and QFQ-banded metaphases specifically for primary constrictions revealed 15 (15%) containing a tdic chromosome with a single primary constriction. Among these chromosomes, 8 (8%) were at the chromosome 15 centromere and 7 (7%) were at the chromosome 5 centromere. The remaining 83 (85%) tdic chromosomes showed two primary constrictions. We analyzed a total of 172 metaphases from peripheral blood, and all except 3 (1.7%) contained the tdic chromosome. Among these three cells, the tdic chromosome was broken in two and absent in one, which indicates that there was some unstable separation of this dicentric in cell division. In two metaphases, there was a chromatid gap at the site of one centromere. Possibly, the absence of certain primary constrictions was associated with deletion of centromeres. This mechanism may be a continual source for additional centromere inactivation during the life of this patient. This case demonstrates that for some dicentrics either centromere may become nonfunctional and inactivation can occur more than once within an individual. The karyotype of this patient was 45,XX,tdic(5;15)(p31;p11). Thus, she was monosomic for about 3/4 of the chromosome 5 short arm. Clinically, this infant had a shrill catlike cry and facies of the cri du chat syndrome.     

A simple model explains the cell cycle-dependent assembly of centromeric nucleosomes in holocentric species

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.     

A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae

We have developed an assay in S. cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes. We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements. Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division. We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III. Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules. Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule. These results suggest plausible pathways for changes in chromosome number that accompany evolution.     

Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.     

Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid

We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis. Received: 30 October 1996 / Accepted: 28 February 1997     

Kinetochore development in two dicentric chromosomes in man

Summary Two dicentric human chromosomes were investigated with light and electron microscopic techniques. One chromosome, with a translocation tdic(5;13)(p12;p12), behaved as a dicentric in about half the cells: it had two primary constrictions; C- and Cd-banding showed two centromeres; and the CREST antikinetochore antibody reacted with the two centromeres with equal affinity. Electron microscopic analysis of sectioned metaphases showed that the dicentric could develop kinetochores at both centromeres simultaneously. The other dicentric chromosome, tdic(21;21)(q22;q22), occasionally showed two primary constrictions, but both C-and Cd-banding distinguished between an active and an inactive centromere, and the CREST antibody reacted only weakly with the inactive centromere. Electron microscopy showed kinetochore development at only one centromere.     

A role for histone H4K16 hypoacetylation in Saccharomyces cerevisiae kinetochore function

Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.     

Multiple satellite deoxyribonucleic acids in the calf and their relation to the sex chromosomes

Four distinct nuclear satellite DNAs from calf (Bos taurus) were isolated and the physical properties of native, single-stranded and renatured duplex molecules of each of the four satellite DNAs were studied by buoyant density-gradient centrifugation. These DNAs were localized in the calf nucleus and on calf metaphase chromosomes by in situ hybridization. In all cases, the calf satellite DNAs are preferentially situated at the centromeres of the autosomes, whereas the X and Y sex chromosomes contain little or none of the satellite DNAs. C-banding techniques showed constitutive heterochromatin at the centromeres of all the autosomes, but not on the X and Y chromosomes.Calf satellite 1 DNA (p = 1.716 g/ml) is at the centromeres of all of the autosomes. Although calf satellite II DNA (p = 1.722 g/ml) is the most widely dispersed over the karyotype, two-thirds of the grains were over the autosomal centromeres. Calf satellites III (p = 1.706 g/ml) and IV (p = 1.709 g/ml) are localized at the centromeres of most, but not all, of the autosomes. The four satellite DNAs each showed a strongly clumped distribution in interphase nuclei of both confluent and growing calf kidney cells in vitro.     

The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.     

Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system

The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such ”Pushmi-Pullyu” hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from ∼2 Mb to ∼20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with ∼2–3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with ∼14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres. Received: 15 December 1998; in revised form: 2 March 1999 / Accepted: 5 April 1999     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

Construction of midget chromosomes in wheat.

To test the usefulness of breakage-fusion-bridge (BFB) cycles in generating new chromosome aberrations in bread wheat (Triticum aestivum L.) and to extend the range of aberrations available, a series of midget chromosomes was produced from the long arm of chromosome 1B. Using a reverse tandem duplication initiated chromatid type BFB cycle, the 1BL arm was broken and fused with centromeres of either chromosome 5BL or 1RS to form dicentric chromosomes. The 1R and 5B centromeres were broken by centric misdivision. Among the progenies of plants with dicentric chromosomes, two classes of monocentric chromosomes were selected: deficient chromosomes 1B and chromosomes that had 1RS or 5BL for one arm and various fragments of 1BL for the other arm. Following centric misdivision of these monocentrics, midget chromosomes 1BL were isolated: deficient and deletion telocentrics and telocentrics derived from interstitial regions of 1BL. By chance, one deficient chromosome 1BS and one deletion chromosome 1BS were identified in unrelated lines of the same wheat. Following centric misdivision of these chromosomes, two midget chromosomes covering the whole of 1BS were added to the set.