Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids

Genomic in situ hybridization (GISH) methods were used to detect different genome components within Brassica amphidiploid species and to identify donor chromatin in hybrids between Brassica napus and Raphanus sativus. In Brassica juncea and Brassica carinata the respective diploid donor genomes could be reliably distinguished by GISH, as could all R-genome chromosomes in the intergeneric hybrids. The A- and C-genome components in B. napus could not be clearly distinguished from one another using GISH, confirming the considerable homoeology between these genomes. GISH methods will be extremely beneficial for monitoring chromatin transfer and introgression in interspecific Brassica hybrids. Received: 20 May 1997 / Accepted: 28 July 1997     

Genomic in situ hybridization in Avena sativa.

Genomic fluorescent in situ hybridization was employed in the study of the genome organization and evolution of hexaploid oat (Avena sativa L. cv. Sun II, AACCDD, 2n = 6x = 42). Genomic DNAs from two diploid oat species, Avena strigosa (genomic constitution AsAs, 2n = 14) and Avena pilosa (genomic constitution CpCp, 2n = 14), were used as probes in the study. The DNA from A. strigosa labelled 28 of the 42 (2/3) chromosomes of the hexaploid oat, while 14 of the 42 (1/3) chromosomes were labelled with A. pilosa DNA, indicating a close relationship between the A and D genomes. Results also suggested that at least 18 chromosomes (9 pairs) were involved in intergenomic interchanges between the A and C genomes.     

Genomic instability in Solanum tuberosum x Solanum kurtzianum interspecific hybrids.

The use of interspecific crosses in breeding is an important strategy in improving the genetic base of the modern cultivated potato, Solanum tuberosum L. Until now, it has normally been interspecific Solanum hybrids that have been morphologically and cytologically characterized. However, little is known about the genomic changes that may occur in the hybrid nucleus owing to the combination of genomes of different origin. We have observed novel AFLP bands in Solanum tuberosum x Solanum kurtzianum diploid hybrids; 40 novel fragments were detected out of 138 AFLP fragments analyzed. No cytological abnormalities were observed in the hybrids; however, we found DNA methylation changes that could be the cause of the observed genomic instabilities. Of 277 MSAP fragments analyzed, 14% showed methylation patterns that differed between the parental species and the hybrids. We also observed frequent methylation changes in the BC1 progeny. Variation patterns among F1 and BC1 plants suggest that some methylation changes occurred at random. The changes observed may have implications for potato breeding as an additional source of variability.     

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.     

Establishment of a multi-color genomic in situ hybridization technique to simultaneously discriminate the three interspecific hybrid genomes in gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

Genomic reorganization and disrupted chromosomal synteny in the siamang (Hylobates syndactylus) revealed by fluorescence in situ hybridization

We employed in situ hybridization (“chromosome painting”) of chromosome-specific DNA libraries of all human chromosomes to establish homologies between the human and siamang karyotypes (Hylobates syndactylus, 2n = 50). Numerous intra- and interchromosomal rearrangements have led to a massive reorganization of the siamang karyotype. There have been a minimum of 33 translocations. The 24 siamang autosomes are composed of 60 recognizable segments that show DNA homology to regions of the 22 human autosomes. Only two autosomes have not been involved in translocations. The siamang presents a case, in a primate closely related to humans, in which chromosome morphology and synteny are highly disturbed in a manner similar to that encountered among rodents. © 1995 Wiley-Liss, Inc.     

Cyto-evolution of Boronia genomes revealed by fluorescent in situ hybridization with rDNA probes.

The physical location of the 25S-26S rDNA sequences was examined in 11 taxa of nine species of Boronia. In diploid species, two rDNA sites were detected in Boronia clavata (2n = 14), Boronia pinnata 'White' (2n = 22), and Boronia chartacea (2n = 32); four in Boronia megastigma (2n = 14) and Boronia denticulata (2n = 18); six in Boronia pinnata 'Pink' (2n = 22); and eight in Boronia molloyae (2n = 16). Eleven sites were found in Boronia heterophylla 'Red' and 'Near White' (2n = 15), but only two active nucleolar organizer regions (NORs) were observed. In polyploid species, Boronia pilosa (2n = 44) had four rDNA sites, while Boronia coerulescens (2n = 72) had six. Most of the rDNA sequences were terminal, but a few were interstitial. There were also differences in signal intensity indicating that the gene copies between and within rDNA sites might be different. The result suggests that considerable chromosome rearrangements have occurred during Boronia cyto-evolution, leading to variation among Boronia taxa in rDNA copy number, site number, and location. These changes together with dysploid reduction during cyto-evolution have made the Boronia genome considerably diverse in chromosome number, genome organization, and chromosome structure.     

Genomic instability in interspecific cell hybrids III. Repression of Colcemid resistance in hybrids suggests preferential β-tubulin expression

A Colcemid-resistant Chinese hamster line with an altered form of β-tubulin was used in studies of the expression of spindle proteins in interspecific cell hybrids. Eight hybrids between this line, and a Colcemid-sensitive mouse cell line, were studied. The altered hamster β-tubulin was not expressed as an increased resistance to Colcemid in any hybrid. Since the complete hamster chromosome complement was represented among the hybrids, the absence of altered β-tubulin is not due to segregation of the mutant hamster β-tubulin gene. We suggest either that the hamster β-tubulin gene is repressed in hybrids, or that hamster β-tubulin is excluded from the spindle in hybrid cells. We compare these findings with previous reports of the repression of other highly active, moderately repeated constitutive genes in interspecific hybrids.     

Detection of specific chromosome reduction in rice somatic hybrids with the A, B, and C genomes by multi-color genomic in situ hybridization

 A multi-color genomic in situ hybridization (McGISH) method has been developed. Three different rice genomes, A, B and C, involved in rice somatic hybrids were distinguished using three different fluorescent signals. All the rice chromosomes from the different genomes could be identified by different fluorescent colors, and the distribution of each genome in the nucleus was clearly visualized under a fluorescence microscope. The relationship between chromosomal constitution and morphological variations observed in the somatic hybrids, and the utility of McGISH, are discussed based on the results currently obtained. Received: 21 November 1997 / Accepted: 9 December 1997     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

Insect-resistant plants with improved horticultural traits from interspecific potato hybrids grown in vitro

Summary Plants were regenerated from petiole calli of interspecific hybrids of Solanum tuberosum x S. berthaultii, an insect-resistant wild species. Callus culture was used to generate genetic changes to overcome the restricted recombination between the two genomes. Two plants out of 58 (3.5%) from calli of hybrid J114-1 showed stable and heritable differences from the hybrid over two cycles of evaluations in the field. Replicated trials were conducted in 1987 and 1988, using two populations of plants propagated by nodal cuttings from the original regenerates maintained in vitro. One regenerate showed insect resistance and increased marketable yield (approximately two fold) in the field. The other had higher levels of phenolic exudate in one of the two types of foliar trichomes associated with the insect resistance mechanism. Some desirable changes were discernible only in sexual progeny of regenerates, not in the regenerates themselves. In a backcross to S. tuberosum, 7 of 14 (50%) regenerates from hybrid F743-4 showed more progeny (up to 15-fold) with improved trichome traits and horticultural characteristics than the original hybrid. The variations were not associated with changes in ploidy. Fifteen plants obtained from these crosses are currently being incorporated into breeding lines. These results suggest that a period of callus culture followed by plant regeneration may aid in the introgression of desirable traits from wild species into crop plants.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

Ultraconserved elements between the genomes of the plants Arabidopsis thaliana and rice

Ultraconserved elements, sequences with 100% identity with no insertions or deletions between genomes, have been found in both vertebrate and invertebrate genomes; whether plant genomes contain ultraconserved elements, however, is unknown. We consequently compared the genomes of Arabidopsis thaliana and rice, which diverged about 200 million years ago, and identified 25 ultraconserved elements that are longer than 100 bp. Similar to those previously found, ultraconserved elements in plants tend to occur in clusters and locate at noncoding regions; nevertheless, they have many distinct features. For instance, the longest ultraconserved element between the 2 plant genomes is 1491 bp, much longer than the longest one (779 bp) between the human and rodent genomes. Some biological implications are discussed, but the functions of these plant ultraconserved elements and the reasons why they are practically frozen during the evolution of millions of years remain a mystery.     

芦荟属植物种间杂交及其F_1代POD同工酶鉴定

以芦荟属植物元江芦荟和皂质芦荟为亲本进行远缘有性杂交试验。正交对3株103朵小花进行授粉,获得7枚果实,平均结实率为6.80%;反交对2株88朵小花授粉,获得5枚果实,平均结实率为5.88%。获得了19株F1代实生苗,并对亲本和F1代实生苗以及库拉索芦荟、华芦荟等5种芦荟属植物进行了过氧化物酶同工酶(POD)的比较研究,结果显示,5种芦荟种间的POD同工酶酶谱具有较高的相似程度,同时各种又具有各自的特征酶带,很容易区分。特别是F1代实生苗的酶谱与父母本的酶谱有显著不同的酶带,证明产生了新的中间类型。     

Genomic in situ hybridization analysis of a trigenomic hybrid involving Solanum and Lycopersicon species.

A 4x potato (+) tomato fusion hybrid (2n = 4x = 48) was successfully backcrossed with a diploid Lycopersicon pennellii (2n = 2x = 24). Genomic in situ hybridization (GISH) on somatic and meiotic chromosomes confirmed that the progenies were triploids (2n = 3x = 36) and possessed three different genomes: potato, tomato, and L. pennellii. Therefore, they have been called trigenomic hybrids. Total genomic probes of both Lycopersicon species were found to hybridize mutually, whereas the potato genome was clearly differentiated. During metaphase I, bivalents were formed predominantly between tomato and L. pennellii chromosomes and the univalents of potato chromosomes were most common. Trivalents in all cases included homoeologous chromosomes of potato, tomato, and L. pennellii. However, the triploids were totally sterile as determined from extensive crossing. On chromosome doubling of triploids by shoot regeneration from callus, hexaploids (2n = 6x = 72) were obtained. Despite exhibiting clear allohexaploid behaviour by forming 36 bivalents at meiosis, these were also completely sterile like their triploid counterparts. In spite of this drawback, the prospects of chromosome pairing between potato L. pennellii and Solanum genomes does open the possibilities for bringing the two genera close.     

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.     

Genomic origin and organization of the allopolyploid Primula egaliksensis investigated by in situ hybridization

Background and Aims: Earlier studies have suggested that the tetraploid Primula egaliksensis(2n = 40) originated from hybridization between the diploidsP. mistassinica (2n = 18) and P. nutans (2n = 22), which werehypothesized to be the maternal and paternal parent, respectively.The present paper is aimed at verifying the hybrid nature ofP. egaliksensis using cytogenetic tools, and to investigatethe extent to which the parental genomes have undergone genomicreorganization. Methods: Genomic in situ hybridization (GISH) and fluorescent in situhybridization (FISH) with ribosomal DNA (rDNA) probes, togetherwith sequencing of the internal transcribed spacer (ITS) regionof the rDNA, were used to identify the origin of P. egaliksensisand to explore its genomic organization, particularly at rDNAloci. Key Results: GISH showed that P. egaliksensis inherited all chromosomes fromP. mistassinica and P. nutans and did not reveal major intergenomicrearrangements between the parental genomes (e.g. interchromosomaltranslocations). However, karyological comparisons and FISHexperiments suggested small-scale rearrangements, particularlyat rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromaticknobs characteristic of the maternal parent P. mistassinicaand maintained only the rDNA loci of P. nutans. These resultscorroborated sequence data indicating that most ITS sequencesof P. egaliksensis were of the paternal repeat type. Conclusions: The lack of major rearrangements may be a consequence of theconsiderable genetic divergence between the putative parents,while the rapid elimination of the ITS repeats from the maternalprogenitor may be explained by the subterminal location of ITSloci or a potential role of nucleolar dominance in chromosomestabilization. These small-scale rearrangements may be indicativeof genome diploidization, but further investigations are neededto confirm this assumption.     

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.