Tracing B-genome chromatin in Brassica napus x B. juncea interspecific progeny.

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny. The B-genome--specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome--positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC(1) progeny. Four B-genome--positive F(2)-derived families and 1 BC(1)-derived family were fixed or segregating for B. juncea morphology in the F(4) and BC(1)S(2), respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F(4)-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome--positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.     

Stable progeny production of the amphidiploid resynthesized Brassica napus cv. Hanakkori, a newly bred vegetable

Resynthesized Brassica napus cv. Hanakkori (AACC, 2n?=?38) was produced by cross-hybridization between B. rapa (AA, 2n?=?20) and B. oleracea (CC, 2n?=?18) as a new vegetative crop. Many studies have provided evidences for the instability and close relationship between A and C genome in the resynthesized B. napus cultivars. In fact, seed produced to obtain progeny in Hanakkori had unstable morphological characters and generated many off-type plants. In this study, we investigated the pollen fertility, chromosome number, structure, and behavior linked to various Hanakkori phenotypes to define factors of unstable phenotypic expression in the progeny. Hanakkori phenotypes were categorized into five types. The results of pollen fertility, chromosome number, and fluorescence in situ hybridization analysis for somatic mitosis cells indicated that the off-type plants had lower pollen fertility, aberrant chromosome number, and structures with small chromosome fragments. Observation of chromosomes at meiosis showed that the meiotic division in off-type plants led to appreciably higher abnormalities than in on-type plants. However, polyvalent chromosomes were observed frequently in both on- and off-type plants in diplotene stage of meiosis. We assume that the unstable morphological characters in resynthesized progeny were the result of abnormal division in meiosis. It results as important that the plants of normal phenotype, chromosome structure and minimized abnormal meiosis are selected to stabilize progeny.     

Introduction of d-genome chromosomes from Aegilops squarrosa L. into tetraploid triticale (AB)(AB)RR (2n=28)

Summary Tetraploid triticale with the genome constitution (ABD) (ABD)RR (2n=4x=28) selected from the progenies of DDRR x (AB)(AB)RR hybrids (D(AB)RR) were karyotyped using C-banding. The aneuploidy frequency was 10.7% with 4.4% hypoploids and 6.3% hyperploids in the F₅. Among 67 plants having 28 chromosomes, 41.8% had a stabilized karyotype, while 58.2% were unstabilized with at least one homoeologous group segregating for A-, B- or D-genome chromosomes. The stabilized plants represented ten different karyotypes that contained one to five disome substitutions of D-genome chromosomes for A- or B-genome chromosomes. Two (BD) (BD)RR tetraploids had no A-genome chromosomes. The average number of D substitutions was 3.0 per line. Of the seven substitutions possible only one, 4D(4B), was not present. In the progeny of plants selected for fertility a selection pressure acted against wheat chromosomes 1B, 3B, 4D and 7D. The most favoured chromosome constitution of the (ABD) mixed genome was 1D, 2A, 3D, 4B, 5B, 6A and 7B. Plants of that karyotype but with a heterologous pair of chromosomes 5B and 5D had the best seed set. Evolutionary and breeding aspects of tetraploid triticale are discussed.     

High efficiency production and genomic in situ hybridization analysis of Brassica aneuploids and homozygous plants

Inplantbreedingandgeneticresearch,karyotypicallystablecrosseswhichproducehybridplantshavebeenextensivelyusedtointroduceintocropsthetargettraitsandgenesfromrelatedwildorcultivatedspeciesortoconstructstocksforgeneticanalysis(alienchromosomeadditions,substitutionsandtranslocations)[1—3].Uniparentalgenomeeliminationinkaryotypicallyunstablehybridshasbeenutilizedforhaploidproduction[2,4].Becausetheartificiallysynthesizedallopoly-ploidscannotbeusedascropsformanyreasons,onepurposeofwidehybridizations…     

High efficiency production and genomic<Emphasis Type="Italic">in situ</Emphasis> hybridization analysis of<Emphasis Type="Italic">Brassica</Emphasis> aneuploids and homozygous plants

Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36-42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30-36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1-4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29-34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1-3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromosome complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.     

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.     

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.     

Inheritance of Arabidopsis DNA in offspring from Brassica napus and A. thaliana somatic hybrids

 Chromosome counts and RFLP markers mapped to Arabidopsis thaliana were used to determine the proportion of eliminated chromosomes and retained A. thaliana DNA in the back-crossed (BC) progeny derived from symmetric and asymmetric somatic hybrids between Brassica napus and A. thaliana. All plants were analysed for the presence of two RFLP markers per chromosome, preferably with one located on each chromosome arm. A reduction in both A. thaliana RFLP markers and chromosome numbers was found in the BC₁ and BC₂ generations of the symmetric hybrids as well as in the BC₁ generation of the asymmetric hybrids. In the symmetric hybrids, two back-crosses to B. napus were required to reduce the frequency of retained A. thaliana loci to 42.4% and mean chromosome number to 39.4. In comparison, the BC₁ progeny of the asymmetric hybrids had 16% of the analysed A. thaliana loci present and an average of 38.4 chromosomes maintained. When the frequency of A. thaliana chromosomes with both analysed loci maintained was compared with the frequency of chromosomes with one locus lost and one kept, a reduction in the number of complete chromosomes between BC₁ and BC₂ derived from the symmetric hybrids was observed. Among the BC₁ plants in the asymmetric group the situation was different, with higher amounts of incomplete donor chromosomes compared to whole chromosomes. The results indicate that A. thaliana chromosome fragments are more often found in the progeny of irradiated hybrids, while back-crossed symmetric hybrids have more complete chromosomes. Received: 2 April 1998 / Accepted: 14 July 1998     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

Production of n + 1 plants and tetrasomics by means of anther culture of trisomic plants in rice (Oryza sativa L.)

Summary Eleven primary trisomics of rice, variety Nipponbare, were subjected to anther culture. The 12th trisomic did not produce normal anthers. A total of 3,734 plants were obtained, which were examined morphologically at the seedling stage in the greenhouse. A number of plants appeared in the progenies of ten trisomics which had unique morphological features. The frequency of these variant types differed among different progenies. Cytological observations revealed that 43 variant plants in the progenies of nine trisomics had 13 chromosomes (n + 1), and 56 were tetrasomics (2n = 26). The tetrasomic plants in the progeny of a trisomic were morphologically identical. Similarly, n + 1 plants in the progeny of a trisomic were also identical. Plants with 23, 25, 36, 39, and 73 chromosomes were also obtained. Results show that valuable aneuploids such as n + 1 and 2n + 2 can be obtained in the anther-culture-derived progenies of trisomics.     

The parasitic effects of rye B chromosomes might be beneficial in the long term

Rye B chromosomes (Bs) have strong parasitic effects on fertility. B carrying plants are less fertile than 0B ones, whereas the Bs have no significant effects on plant vigour. On the other hand, it has been reported that B transmission is under genetic control in such a way that H line plants transmit the Bs at high frequency, whereas the Bs in the low B transmission rate line (L) fail to pair at metaphase I and are frequently lost. In the present work we analyse variables affecting vigour and fertility considering not only the number of Bs of each plant, but also its H or L status and the B number of its maternal parent. Our results show that the Bs not only decrease female fertility of the B carrier, but the fertility of its progeny, with the exception of 0B plants coming from a 4B mother, which are the most fertile. In this way B chromosomes can be considered as a selective factor. Pollen abortion was higher in B carriers, in the progeny of B carriers and in H plants, but 4B plants coming from B carrying mothers produce less aborted pollen, indicating that a high B number is more deleterious if it is transmitted in the pollen grains. A similar result was obtained for endosperm quality estimated as grain weight, because it is negatively influenced by the Bs in 4B plants coming from a 0B mother. H plants were always less fertile than L ones, indicating that alleles increasing the loss of Bs in the L line will be probably selected as a defence of the A genome against the invasive Bs of the H line. Flower number is not affected by the Bs.     

Effect of substituting chromosome 5A with an supernumary chromosome 5B in offspring of a somaclonal common wheat line monosomic for 5A

Cytogenetic analysis was employed in studying the cause of generation of fertile awned forms in the progeny of plants, which were selected from a speltoid somaclonal wheat line monosomic for chromosome 5A (2n = 41 = 20II + I), had speltoid spikes, and were reproduced by self pollination. On cytogenetic and genetic evidence, chromosome 5A was eliminated and the copy number of chromosome 5B increased in the plants examined. The appearance of an extra chromosome 5B is probably caused by nondisjunction of bivalent 5B in the presence of a telocentric originating from the long arm of chromosome 5A. A difference in meiotic segregation was observed for univalent chromosomes 5A and 5B.     

The effect of B chromosomes on fitness components inAegilops speltoides Tausch

The effect of B chromosomes (Bs) on various components of vigour and fertility inAegilops speltoides was studied. We designed three types of synthetic populations. In the first type we compared plants from the localities Haifa (which has Bs in nature) and Ashkelon (which has not). Non significant differences were found between the plants from both populations, and between B carriers and non carriers among Haifa individuals. In the second synthetic population we compared 0B, 1B, 2B, 3B and 5B plants from Haifa. We found that vigour variables were not affected by the presence of Bs, while fertility variables were significantly correlated with B number, in such a way that 1B plants produce more grains per plant than any other constitution, while 5Bs produce a remarkable deleterious effect. In the third type of synthetic populations we compared plants with 0B and 1B, 0B and 2B, 0B and 3B, and 0B and 5B. In this case we also found that 1B plants were the most fertile, but the variation of reproductive fitness was higher from plot to plot within the same B class than among B classes. We conclude that the effect of B chromosomes on the fitness variables that we have estimated is close to neutrality in this species. We discuss the relation between the strength of the mechanism of accumulation and the deleterious effects of B chromosomes.     

PCR and sequence analysis of barley chromosome 2H subjected to the gametocidal action of chromosome 2C

Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.     

The Effect of Substitution of Chromosome 5A with an Extra Chromosome 5B in the Progeny of a Somaclonal Common Wheat Line Monosomic for 5A

Cytogenetic analysis was employed in studying the cause of generation of fertile awned forms in the progeny of plants, which were selected from a speltoid somaclonal wheat line monosomic for chromosome 5A (2n = 41 = 20II + I), had speltoid spikes, and were reproduced by self pollination. On cytogenetic and genetic evidence, chromosome 5A was eliminated and the copy number of chromosome 5B increased in the plants examined. The appearance of an extra chromosome 5B is probably caused by nondisjunction of bivalent 5B in the presence of a telocentric originating from the long arm of chromosome 5A. A difference in meiotic segregation was observed for univalent chromosomes 5A and 5B.     

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.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line “TAI-27” revealed by fluorescencein situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line "TAI-27"revealed by fluorescence in situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.     

斑茅割手密复合体(GXAS07-6-1)及其与甘蔗F_1的GISH分析

斑茅割手密复合体(GXAS07-6-1)是广西蔗茅属斑茅和广西甘蔗属割手密的属间杂种,聚集了双亲的优点。本研究利用基于Alu-like的PCR鉴定方法对GXAS07-6-1及甘蔗与GXAS07-6-1的3份F_1材料进行真实性鉴定,基于基因组原位杂交技术(GISH)对父本GXAS07-6-1及其3份F_1染色体组成及核型进行分析。研究结果表明:3份F_1材料为GXAS07-6-1的真杂种;父本GXAS07-6-1的染色体众数为62条,其中30条来自蔗茅属斑茅,32条来自甘蔗属割手密,核型分类属于1B,其染色体按"n+n"方式传递;GXASF_108-2-17、GXASF_108-2-22、GXASF_108-2-32的染色体数目为78~80条,其中69~71条来自甘蔗属,9~11条来自蔗茅属斑茅,3份F_1的核型分类分别属于2B、1B、1B,染色体传递方式均为"n+n"。父本GXAS07-6-1及3份F_1材料中均未发现有染色体的交换或易位现象。甘蔗与斑割复合体杂交,蔗茅属斑茅染色体在亲子间传递过程存在丢失现象。