Transmission of alien chromosomes from selfed progenies of a complete set of Allium monosomic additions: the development of a reliable method for the maintenance of a monosomic addition set.

Selfed progeny of a complete set of Allium fistulosum - Allium cepa monosomic addition lines (2n = 2x + 1 = 17, FF+1A-FF+8A) were produced to examine the transmission rates of respective alien chromosomes. All eight types of the selfed monosomic additions set germinable seeds. The numbers of chromosomes (2n) in the seedlings were 16, 17, or 18. The eight extra chromosomes varied in transmission rate (%) from 9 (FF+2A) to 49 (FF+8A). The complete set of monosomic additions was reproduced successfully by self-pollination. A reliable way to maintain a set of Allium monosomic additions was developed using a combination of two crossing methods, selfing and female transmission. FF+8A produced two seedlings with 18 chromosomes. Cytogenetical analyses, including GISH, showed that the seedlings were disomic addition plants carrying two entire homologous chromosomes from A. cepa in an integral diploid background of A. fistulosum. Flow cytometry analysis showed that a double dose of the alien 8A chromosome caused fluorescence intensity values spurring in DNA content, and isozyme analysis showed increased glutamate dehydrogenase activity at the gene locus Gdh-1.     

Complete assignment of structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of the shallot (Allium cepa L.)

We analyzed Japanese bunching onion (Allium fistulosum L.) - shallot (Allium cepa L. Aggregatum group) alien chromosome addition lines in order to assign the genes involved in the flavonoid biosynthesis pathway to chromosomes of the shallot. Two complete sets of alien monosomic additions (2n = 2x + 1 = 17) were used for determining the chromosomal locations of several partial sequences of candidate genes, CHS, CHI, F3H, DFR, and ANS via analyses of PCR-based markers. The results of DNA marker analyses showed that the CHS-A, CHS-B, CHI, F3H, DFR, and ANS genes should be assigned to chromosomes 2A, 4A, 3A, 3A, 7A, and 4A, respectively. HPLC analyses of 14 A. fistulosum - shallot multiple alien additions (2n = 2x + 2 - 2x + 7 = 18 - 23) were conducted to identify the anthocyanin compounds produced in the scaly leaves. A direct comparison between the genomic constitution and the anthocyanin compositions of the multiple additions revealed that a 3GT gene for glucosylation of anthocyanidin was located on 4A. Thus, we were able to assign all structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of A. cepa.     

Construction of SSR-based chromosome map in bunching onion (Allium fistulosum)

We have constructed a linkage map of bunching onion (Allium fistulosum L., 2n = 16) using an F(2) population of 225 plants. The map consists of 17 linkage groups with 212 bunching onion SSR markers and 42 bulb onion (A. cepa L.) SSR, InDel, CAPS or dCAPS markers, covering 2,069 cM. This is the first report of a linkage map mainly based on SSR markers in the genus Allium. With the 103 anchor markers [81 bunching onion SSRs, 11 bulb onion SSRs and 11 bulb onion non-SSRs (1 InDel, 9 CAPSs and 1 dCAPS)] whose chromosome assignments were identified in A. cepa and/or A. fistulosum, via the use of several kinds of Allium alien addition lines, 16 of the 17 linkage groups were connected to the 8 basic chromosomes of A. cepa.     

Effect of single alien chromosome from shallot (Allium cepa L. Aggregatum group) on carbohydrate production in leaf blade of bunching onion (A. fistulosum L.)

We used a complete set of Allium fistulosum - shallot (A. cepa Aggregatum group) monosomic addition lines (FF+1A - FF+8A) to identify shallot chromosomes affecting the production of sugars. In the alien addition lines grown over two years in an experimental field at Yamaguchi University (34 degrees N, 131 degrees E), shallot chromosomes 2A and 8A altered sugar contents in leaf-bunching onion (A. fistulosum). Except for FF+2A, every monosomic addition accumulated non-reducing sugars in winter leaf blades. FF+8A caused an increase in the amounts of non-reducing sugars in the winter. FF+2A hardly produced non-reducing sugar throughout the two-year study. These results indicated that genes related to non-reducing sugar metabolism are located on the 2A and 8A chromosomes. The results of regression analyses using 2002 data on A. fistulosum and the monosomic addition set revealed a correlation (r = 0.63 +/- 0.07; mean +/- SE., n = 9) between reducing sugar and monosaccharide (Glc+Fru) contents but no correlation between non-reducing sugar and sucrose contents. This result indicates the existence of other polysaccharides (e.g., scorodose) as non-reducing sugars in the leaf blade.     

Production and characterization of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of Japanese bunching onion (A. fistulosum L.)

First and second backcrosses of amphidiploid hybrids (2n = 4x = 32, genomes AAFF) between shallot (Allium cepa Aggregatum group) and A. fistulosum were conducted to produce A. cepa - A. fistulosum alien addition lines. When shallot (A. cepa Aggregatum group) was used as a pollinator, the amphidiploids and allotriploids set germinable BC(1) and BC(2) seeds, respectively. The 237 BC(1) plants mainly consisted of 170 allotriploids (2n = 3x = 24, AAF) and 42 hypo-allotriploids possessing 23 chromosomes, i.e., single-alien deletions (2n = 3x-1 = 23, AAF-nF). The single-alien deletions in the BC(1) progeny showed dwarfing characteristics and were discriminated from the allotriploids (2n = 24) and hyper-allotriploids (2n = 25) by means of flow cytometric analysis. The chromosome numbers of 46 BC(2) seedlings varied from 16 to 24. Eight monosomic additions (2n = 2x+1 = 17, AA+nF) and 20 single-alien deletions were found in these BC(2) seedlings. Consequently, six kinds of A. cepa - A. fistulosum alien chromosome additions possessing different chromosome numbers (2n = 17, 18, 20, 21, 22, 23) were recognized in the BC(1) and BC(2) populations. A total of 79 aneuploids, including 62 single-alien deletions, were analyzed by a chromosome 6F-specific isozyme marker (Got-2) in order to recognize its existence in their chromosome complements. This analysis revealed that two out of 62 single-alien deletions did not possess 6F. One (AAF-6F) out of the possible eight single-alien deletions could be identified at first. The present study is a first step toward the development of a useful tool, such as a complete set of eight different single-alien deletions, for the rapid chromosomal assignment of genes and genetic markers in A. fistulosum.     

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei. Received: 16 April 1999 / Accepted: 13 August 1999     

Biochemical and genetic analysis of carbohydrate accumulation in Allium cepa L

Onion and shallot (Allium cepa L.) exhibit wide variation in bulb fructan content, and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc, we conducted biochemical and genetic analyses of Allium fistulosum (FF)-shallot (A. cepa Aggregatum group) alien monosomic addition lines (AALs; FF+1A-FF+8A) and onion mapping populations. Sucrose and fructan levels in leaves of FF+2A were significantly lower than in FF throughout the year, and the springtime activity of acid invertase was also lower. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Inbred high fructan (Frc_) lines from the 'W202Ax Texas Grano 438' onion population exhibited significantly higher sucrose levels prior to bulbing than low fructan (frcfrc) lines. Sucrose synthase (SuSy) activity in these lines was correlated with leaf hexose content but not with Frc phenotype. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion expressed sequence tag resources. SPS and SuSy loci were assigned to chromosome 8 and 6, respectively, using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and three invertases) and 8 (neutral invertase). The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum suggest that the Frc locus may condition variation in SPS activity.     

The identification and analysis of the sequences that allow the detection of <Emphasis Type="Italic">Allium cepa</Emphasis> chromosomes by GISH in the allodiploid <Emphasis Type="Italic">A. wakegi</Emphasis>

In Allium wakegi, which is an allodiploid species between Allium cepa and Allium fistulosum, each genome can be clearly distinguished using genomic in situ hybridization (GISH). Genomic DNA of A. cepa and A. fistulosum is differentiated both qualitatively and quantitatively. We wanted to isolate nucleotide sequences that give genome-specific signals on A. cepa chromosomes in GISH experiments in A. wakegi. We isolated 23 clones that show GISH-like signal patterns in fluorescence in situ hybridization (FISH) and analyzed their distribution in the A. cepa- and A. fistulosum-derived genomes of A. wakegi. There was considerable variation in the abundance and distribution of these cloned sequences on the chromosomes of the two species. The degree of A. cepa specificity varied among the clones. Twenty-two of the clones showed an even distribution over most chromosome arms with some clustering in the pericentromeric regions, but one clone showed very distinct terminal signals on some chromosomes. Whereas these sequences are not specific for A. cepa, changes in bases in nucleotide sequences and in their amount result in genome-specific characteristics in GISH experiments.     

Direct comparison between genomic constitution and flavonoid contents in Allium multiple alien addition lines reveals chromosomal locations of genes related to biosynthesis from dihydrokaempferol to quercetin glucosides in scaly leaf of shallot (Allium cepa L.)

The extrachromosome 5A of shallot (Allium cepa L., genomes AA) has an important role in flavonoid biosynthesis in the scaly leaf of Allium fistulosum–shallot monosomic addition lines (FF+nA). This study deals with the production and biochemical characterisation of A. fistulosum–shallot multiple alien addition lines carrying at least 5A to determine the chromosomal locations of genes for quercetin formation. The multiple alien additions were selected from the crossing between allotriploid FFA (♀) and A. fistulosum (♂). The 113 plants obtained from this cross were analysed by a chromosome 5A-specific PGI isozyme marker of shallot. Thirty plants were preliminarily selected for an alien addition carrying 5A. The chromosome numbers of the 30 plants varied from 18 to 23. The other extrachromosomes in 19 plants were completely identified by using seven other chromosome markers of shallot. High-performance liquid chromatography analyses of the 19 multiple additions were conducted to identify the flavonoid compounds produced in the scaly leaves. Direct comparisons between the chromosomal constitution and the flavonoid contents of the multiple alien additions revealed that a flavonoid 3′-hydroxylase (F3′H) gene for the synthesis of quercetin from kaempferol was located on 7A and that an anonymous gene involved in the glucosidation of quercetin was on 3A or 4A. As a result of supplemental SCAR analyses by using genomic DNAs from two complete sets of A. fistulosum–shallot monosomic additions, we have assigned F3′H to 7A and flavonol synthase to 4A.     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

应用基因组原位杂交及RFLP标记鉴定小麦中的大麦染色体

用生物素（Biotin-6-dUTP)标记的大麦Betzes基因组DNA作探针,以普通小麦中国春总DNA作封阻进行基因组原位杂交（Genomeinsituhybridization,简称GISH）,从13株小麦-大麦杂交后代中鉴定出2个含有3条大麦Betzes2H染色体的材料（2n＝43）;2个2H单体异代换系（2n＝42）;7个2H二体异代换系（2n＝42）。用已定位在小麦第2部分同源群短臂上的探针psr131进行RFLP分析,结果表明大麦Betzes、代换系A5有1条区别于小麦中国春的特异带,A     

Organization of the 378 bp satellite repeat in terminal heterochromatin of Allium fistulosum

Telomeres, DNA-protein structures, are important elements of the eukaryotic chromosome. Telomeric regions of the majority of higher plants contain heptanucleotides TTTAGGG arranged into a tandem repeat. However, some taxa have no such repeats. These are some species of lilies (Lilium) and onions (Allium). For example, terminal regions of chromosomes of Spanish onion (Allium fistulosum) contain satellite DNA whose unit repeats are 380 bp in length, and the short arm of its chromosome 8 contains rDNA repeats. This study deals with the terminal heterochromatin and organization of the satellite repeat in A. fistulosum. Fluorescent in situ hybridization (FISH) was used to locate the satellite DNA on chromosomes and on extended DNA of A. fistulosum. Nonsatellite DNA was found in the structure of telomeric repeat. Polymerase chain reaction (PCR) and Southern hybridization were used for analysis of terminal heterochromatin. Various rearrangements were found in the satellite repeat. The roles of retrotransposones and microsatellites in the formation of terminal heterochromatin are discussed.     

Production and cytogenetics of Brassica campestris-alboglabra chromosome addition lines

 Four different Brassica campestris-alboglabra monosomic addition lines (AA+1 chromosome from C, 2n=21) were obtained after consecutive backcrosses between resynthesized B. napus (AACC, 2n=38) and the parental B. campestris (AA, 2n=20) accession. The alien chromosomes of B. alboglabra (CC, 2n=18) in the addition lines were distinguished by random amplified polymorphic DNA (RAPD) marker analysis and morphology of mitotic chromosomes. Four RAPD marker synteny groups were established, which represented the four different alien chromosomes of B. alboglabra in the four addition lines. Three of the four addition lines were identified to harbour chromosomes 4, 8 or 9 of B. alboglabra. Studies on meiotic pairing in the addition lines revealed intergenomic homoeology relationships among specific chromosome arms between the A- and C-genomes. The long arm of B. campestris chromosome 9 was homoeologous with the long arm of B. alboglabra chromosome 4, while its short arm with the short arms of B. alboglabra chromosomes 8 and 9. Such an intergenomic homoeology relationship supports the hypothesis that B. campestris and B. alboglabra share a common ancestor but that chromosomal rearrangements have occurred during the evolution of the two species. Intergenomic introgression was observed in the progenies of the addition lines. The introgression of an entire B. alboglabra marker synteny group into the B. campestris genome implied the possible occurrence of interspecific chromosomal substitution. Received: 30 May 1996 / Accepted: 18 October 1996     

DNA methylation and histone modification in onion chromosomes

Onion, Allium cepa, is a model plant for experimental observation of somatic cell division, whose mitotic chromosome is extremely large, and contains the characteristic terminal heterochromatin. Epigenetic status of the onion chromosome is a matter of deep interest from a molecular cytogenetic point of view, because epigenetic marks regulate chromatin structure and gene expression. Here we examined chromosomal distribution of DNA methylation and histone modification in A. cepa in order to reveal the chromatin structure in detail. Immunodetection of 5-methylcytosine (5mC) and in situ nick-translation analysis showed that onion genomic DNA was highly methylated, and the methylated CG dinucleotides were distributed in entire chromosomes. In addition, distributions of histone methylation codes, which occur in close association with DNA methylation, were similar to those of other large genome species. From these results, a highly heterochromatic and less euchromatic state of large onion chromosomes were demonstrated at an epigenetic level.     

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

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

大白菜-结球甘蓝5号和8号单体异附加系的筛选和鉴定

为建立成套的大白菜-结球甘蓝异附加系,以大白菜-结球甘蓝异源三倍体（AAC,2n=3x=29）与二倍体大白菜（AA,2n=2x=20）回交世代BC1为材料,对其进行细胞学观察并从中筛选单体异附加系。结果表明,BC1群体植株发生了染色体数目变异,变异范围为2n=20～29,其中以2n=23～24的非整倍体植株居多,占总观察株数的51．44％;对2个2n=21的植株进行核型分析,初步鉴定为大白菜-结球甘蓝5号和8号单体异附加系,分别命名为CO-5—1和CO-8—2。     

Development and characterization of potato-Solanum brevidens chromosomal addition/substitution lines

Solanum brevidens is a wild diploid potato species possessing high levels of resistances to several major potato diseases. We previously developed fertile somatic hybrids between S. brevidens and the cultivated potato (Solanum tuberosum) in order to introgress disease resistances from this wild species into potato. A series of backcross progenies was developed from a hexaploid somatic hybrid A206. Using a combination of S. brevidens-specific randomly amplified polymorphic DNA (RAPD) markers and a sequential genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) technique, we identified all 12 S. brevidens chromosomes in the backcross progenies. Seven potato-S. brevidens monosomic chromosome addition lines (chromosomes 1, 3, 4, 5, 8, 9 and 10) and one monosomic substitution line (chromosome 6) were identified, and the remaining four S. brevidens chromosomes (2, 7, 11, and 12) were included in two other lines. These chromosomal addition/substitution stocks provide valuable tools for potato cytogenetic research, and can be used to introgress disease resistances from S. brevidens into potato.     

Production of alien chromosome additions and their utility in plant genetics

Breeding programs aiming at transferring desirable genes from one species to another through interspecific hybridization and backcrossings often produce monosomic and disomic additions as intermediate crossing products. Such aneuploids contain alien chromosomes added to the complements of the recipient parent and can be used for further introgression programs, but lack of homoeologous recombination and inevitable segregation of the alien chromosome at meiosis make them often less ideal for producing stable introgression lines. Monosomic and disomic additions can have specific morphological characteristics, but more often they need additional confirmation of molecular marker analyses and assessment by fluorescence in situ hybridization with genomic and chromosome-specific DNA as probes. Their specific genetic and cytogenetic properties make them powerful tools for fundamental research elucidating regulation of homoeologous recombination, distribution of chromosome-specific markers and repetitive DNA sequences, and regulation of heterologous gene expression. In this overview we present the major characteristics of such interspecific aneuploids highlighting their advantages and drawbacks for breeding and fundamental research.     

Establishment of a complete series of a monosomic tomato chromosome addition lines in the cultivated potato using RFLP and GISH analyses

With the aim of establishing a complete monosomic alien tomato chromosome addition series in a potato background, the backcross progenies derived from repeated crossing of potato (+) tomato fusion hybrids to potato were screened through RFLP and GISH analyses. Because of the availability from our previous work of seven of the possible 12 tomato monosomic additions, selected from BC₂ populations, attention was paid to those alien additions that were missing. Thus, since the alien additions were already available for tomato chromosomes 1, 2, 4, 6, 8, 10 and 12, efforts were made to select for chromosomes 3, 5, 7, 9 and 11 by screening specific BC₃ populations. In all, 105 plants from four BC₃ populations were screened through a combination of RFLP and GISH analyses in order to complete the series. Among the newly selected alien addition lines, five were monosomic additions for all the remaining chromosomes and one was a disomic addition for chromosome 11. When using conventional cytogenetics the selection of monosomic alien additions is highly laborious. All the tomato chromosomes showed a variable rate of transmission. Chromosome 6 was transmitted at 29.6% and 81.5% frequency in populations 2705 and 2701 respectively. The present study showed that molecular markers and molecular cytogenetics applied in this study were most efficient and rapid because a pre-selection for the desired genotypes was possible by screening a population with chromosome-specific markers for the presence of one tomato chromosome at a time. Received: 9 January 2001 / Accepted: 26 January 2001     

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.