Genomic In Situ Hybridization Analysis Research Articles

Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae, Leguminosae), an important legume pasture crop.

The genus Stylosanthes includes nitrogen-fixing and drought-tolerant species of considerable economic importance for perennial pasture, green manure and land recovery. Stylosanthes scabra is adapted to variable soil conditions, being cultivated to improve pastures and soils worldwide. Previous studies have proposed S. scabra as an allotetraploid species (2n = 40) with a putative diploid A genome progenitor S. hamata or S. seabrana (2n = 20) and the B genome progenitor S. viscosa (2n = 20). We aimed to provide conclusive evidence for the origin of S. scabra. We performed fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) experiments and Illumina paired-end sequencing of S. scabra, S. hamata and S. viscosa genomic DNA, to assemble and compare complete ribosomal DNA (rDNA) units and chloroplast genomes. Plastome- and genome-wide single nucleotide variation detection was also performed. GISH and phylogenetic analyses of plastid DNA and rDNA sequences support that S. scabra is an allotetraploid formed from 0.63 to 0.52 million years ago (Mya), from progenitors with a similar genome structure to the maternal donor S. hamata and the paternal donor S. viscosa. FISH revealed a non-additive number of 35S rDNA sites in S. scabra compared with its progenitors, indicating the loss of one locus from A genome origin. In S. scabra, most 5S rDNA units were similar to S. viscosa, while one 5S rDNA site of reduced size most probably came from an A genome species as revealed by GISH and in silico analysis. Our approach combined whole-plastome and rDNA assembly with additional cytogenetic analysis to shed light successfully on the allotetraploid origin of S. scabra. We propose a Middle Pleistocene origin for S. scabra involving species with maternal A and paternal B genomes. Our data also suggest that variation found in rDNA units in S. scabra and its progenitors reveals differences that can be explained by homogenization, deletion and amplification processes that have occurred since its origin.

Production and Molecular Cytogenetic Characterization of a Durum Wheat-Thinopyrum elongatum 7E Disomic Addition Line with Resistance to Fusarium Head Blight

Wheatgrass, Thinopyrum elongatum (2n = 2x = 14, EE), is an important wild relative of wheat with many excellent traits, including resistance to Fusarium head blight (FHB), that can be used for durum wheat improvement. Through hybridization of the durum cultivar “Langdon” with the amphiploid 8801 (AABBEE), a disomic alien addition line (2n = 30) with a pair of Th. elongatum 7E chromosomes was obtained and confirmed using chromosome-specific molecular markers of Th. elongatum and genomic in situ hybridization (GISH). This line is meiotically and reproductively stable, generally forming 15 bivalents at meiosis including 14 pairs from Langdon and 1 from Th. elongatum with 2 chiasmata each as revealed by GISH analysis. At the adult growth stages under field conditions, this addition line shows high resistance to FHB, with less than 16% infection on visual observation in 2 years (2014 and 2015). This addition line is shorter in height and has narrower leaves and shorter spikes as compared to its parent Langdon. So the linkage group 7E might be a further source of wheat improvement by targeted introgression approaches.

Molecular cytogenetic identification of a wheat - Thinopyrum ponticum substitution line with stripe rust resistance.

Thinopyrum ponticum (Th. ponticum) (2n = 10x = 70) is an important breeding material with excellent resistance and stress tolerance. In this study, we characterized the derivative line CH1113-B13-1-1-2-1 (CH1113-B13) through cytological, morphological, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), expressed sequence tag (EST), and PCR-based landmark unique gene (PLUG) marker analysis. The GISH analysis revealed that CH1113-B13 contained 20 pairs of common wheat chromosomes and one pair of JSt genomic chromosomes. Linkage analysis of Th. ponticum using seven EST and seven PLUG markers indicated that the pair of alien chromosomes belonged to the seventh homeologous group. Nulli-tetrasomic and FISH analysis revealed that wheat 7B chromosomes were absent in CH1113-B13; thus, CH1113-B13 was identified as a 7JSt (7B) substitution line. Finally, adult-stage CH1113-B13 exhibited immunity to wheat stripe rust. This substitution line is therefore a promising germplasm resource for wheat breeding.

Molecular cytogenetics identification of a wheat –Leymus mollisdouble disomic addition line with stripe rust resistance

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) possesses a number of valuable genes against biotic and abiotic stress, which could be transferred into common wheat background for wheat improvement. In the present study, we determined the karyotypic constitution of a wheat - L. mollis double disomic addition line, M11003-4-4-1-1, selected from the F5 progeny of a stable wheat - L. mollis derivative M39 (2n = 56) × Triticum aestivum cultivar 7182, by morphological and cytogenetic identification, GISH (genomic in situ hybridization), FISH (fluorescent in situ hybridization), molecular markers analysis, and stripe rust resistance evaluation. Cytological studies demonstrated that M11003-4-4-1-1 had a chromosome karyotype of 2n = 46 with 23 bivalents, while GISH and FISH analysis indicated that this line contained 42 common wheat chromosomes and two pairs of L. mollis chromosomes. DNA markers showed that the alien chromosomes from L. mollis belonged to homoeologous groups 5 and 6. Evaluation of the agronomic traits revealed that M11003-4-4-1-1 was resistant to stripe rust at the adult stage, while the plant height was reduced and the 1000-grain weight was increased significantly. Therefore, the new line M11003-4-4-1-1 could be exploited as an important bridge material in chromosome engineering and wheat breeding.

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH, PCR-RFLP and RFLP.

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.

Insight into octoploid strawberry (Fragaria) subgenome composition revealed by GISH analysis of pentaploid hybrids.

As the product of interspecific hybridization between its two ancestral octoploid (2n = 8x = 56) species (Fragaria chiloensis and F. virginiana), the cultivated strawberry (F. ×ananassa) is among the most genomically complex of crop plants, harboring subgenomic components derived from as many as four different diploid ancestors. To physically visualize the octoploids' subgenome composition(s), we launched molecular cytogenetic studies using genomic in situ hybridization (GISH), comparative GISH (cGISH), and rDNA-FISH techniques. First, GISH resolution in Fragaria was tested by using diploid and triploid hybrids with predetermined genome compositions. Then, observation of an octoploid genome was implemented by hybridizing chromosomes of pentaploid (2n = 5x = 35) hybrids from F. vesca × F. virginiana with genomic DNA probes derived from diploids (2n = 2x = 14) F. vesca and F. iinumae, which have been proposed by phylogenetic studies to be closely related to the octoploids yet highly divergent from each other. GISH and cGISH results indicated that octoploid-derived gametes (n = 4x = 28) carried seven chromosomes with hybridization affinities to F. vesca, while the remaining 21 chromosomes displayed varying affinities to F. iinumae, indicating differing degrees of subgenomic contribution to the octoploids by these two putatively ancestral diploids. Combined rDNA-FISH revealed severe 25S rDNA loss in both the F. vesca- and F. iinumae-like chromosome groups, while only the prior group retained its 5S loci.

Cytological characterization of an interspecific hybrid in <i>Jatropha</i> and its progeny reveals preferential uniparental chromosome transmission and interspecific translocation

Genetic variation in Jatropha curcas, a prospective biodiesel plant, is limited, and interspecific hybridization needed for its genetic improvement. Progeny from interspecific crosses between J. curcas and Jatropha integerrima can be used to improve agronomic characters and to increase oil content and yield. However, these hybrids have not been characterized cytologically. The present study was aimed at the analysis of chromosome behavior during meiosis and chromosome composition of S1 plants derived from an interspecific F1 hybrid using genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Bivalents that formed as a result of interspecific pairing were frequently observed, suggesting the presence of homoeologous chromosomes from the two species. Almost half of microspores were derived from the reduction division; GISH analysis indicated random transmission of the parent chromosomes to microspores. Male fertility measured as pollen staining with acetocarmine was 48.4%. In contrast, GISH analysis of S1 plants revealed preferential transmission of J. curcas chromosomes. We also found segment exchange between chromosomes of the two species (interspecific translocation) by GISH and FISH analyses. Introgression of J. integerrima chromosome segments into the J. curcas genome would help to improve Jatropha cultivars for mass production.

Introgression of Agropyron cristatum 6P chromosome segment into common wheat for enhanced thousand-grain weight and spike length.

This study explored the genetic constitutions of wheat-Agropyron cristatum 6P chromosomal translocation and determined the effects of 6P intercalary chromosome segment on thousand-grain weight and spike length in wheat. Crop wild relatives provide rich genetic resources for wheat improvement. Introduction of alien genes from Agropyron cristatum into common wheat can broaden its genetic diversity. In this study, radiation-induced wheat-A. cristatum translocation line Pubing3035 derived from the offspring of wheat-A. cristatum 6P chromosomes addition line was identified and analyzed using genomic in situ hybridization (GISH), dual-color fluorescence in situ hybridization (FISH), and molecular markers. GISH analysis revealed that Pubing3035 was a Ti1AS-6PL-1AS·1AL intercalary translocation. The breakpoint was pinpointed to locate near the centromeric region on the short arm of wheat chromosome 1A based on a constructed F2 linkage map and it was flanked by markers SSR12 and SSR263. The genotypic data, combined with the phenotypes, indicated that A. cristatum 6P chromosomal segment played an important role in regulating the thousand-grain weight and spike length. On average, the thousand-grain weight and spike length in translocation individuals were approximately 2.5 g higher and 0.7 cm longer than those in non-translocation individuals in F2 and BC1F1 populations. The clusters of quantitative trait loci for thousand-grain weight, spike length, and spikelet density contributed by 6P chromosome segment were mapped between A. cristatum unique marker Agc7155 and wheat marker SSR263, which, respectively, explained phenotypic variance of 24.96, 12.38 and 17.20 % with an LOD of 10.63, 4.89 and 5.59. Overall, the translocation Pubing3035 had a positive effect on the yield of wheat, which laid the foundation for the localization of A. cristatum excellent genes and made itself a promising and valuable germplasm for wheat improvement.

Molecular-cytogenetic analysis of Aegilops triuncialis and identification of its chromosomes in the background of wheat.

BackgroundSpecies belonging to the genus Aegilops L. are an important source of genetic material for expanding genetic variability of wheat. Ae. triuncialis is an allotetraploid in this genus which was originated from hybridization of Ae. umbellulata and Ae. markgrafii (Greuter) Hammer. Although the Ae. triuncialis karyotype was thoroughly examined by conventional chromosome staining and Giemsa C-banding, it is still poorly characterized using FISH markers. The objective of this study was to test the fluorescence in situ hybridization (FISH) patterns of Ae. triuncialis (2n = 4x = 28, CtCtUtUt) chromosomes using different repetitive sequences and to compare the produced patterns to the chromosomes of its diploid ancestors, with the aim of establishing a generalized Ae. triuncialis idiogram and detection of Aegilops chromosomes in the background of wheat.ResultsThe probes pSc119.2-1, pTa535-1, pAs1-1, (CTT)10 and the 45S rDNA clone from wheat (pTa71) were hybridized to chromosomes of Ae. triuncialis and compared with its diploid progenitors (Ae. umbellulata Zhuk., 2n = 2x = 14, UU and Ae. markgrafii (Greuter) Hammer, 2n = 2x = 14, CC) and Ae. cylindrica Host. (2n = 4x = 28, DcDcCcCc), another tetraploid species containing the C-genome. Ae. cylindrica was further analyzed by genomic in situ hybridization (GISH) using C genome probe in order to identify any possible translocation.ConclusionsIn general, FISH patterns of the Ut- and Ct-genome chromosomes of Ae. triuncialis were similar to those of U- and C-genome chromosomes of the diploid progenitor species Ae. umbellulata and Ae. markgrafii respectively, although some differences were observed. Two major 45S rDNA loci were revealed in the short arm of chromosomes A and C, of the Ct genome which correspond to homoeologous groups 1 and 5 respectively. Minor 45S rDNA loci were mapped on the short arm of chromosomes 1Ut and 5Ut. GISH analysis revealed three different non-reciprocal homologous or heterologous translocations between Cc and Dc chromosomes in all studied accessions of Ae. cylindrica.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-014-0091-6) contains supplementary material, which is available to authorized users.

Development and application of PCR markers specific to the 1Ns chromosome of Psathyrostachys huashanica Keng with leaf rust resistance

Wheat–Psathyrostachys huashanica Keng disomic addition line 12-3 was developed and characterized using genomic in situ hybridization (GISH), expressed sequence tag–sequence tagged site (EST–STS), and sequence characterized amplified region (SCAR) markers. Mitotic and meiotic GISH analyses indicated that it contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. Eight EST–STS multiple-loci markers located on the homoeologous group 1 chromosomes of wheat amplified polymorphic bands in the 1Ns disomic addition line 12-3, which were unique to P. huashanica. These markers suggested that the introduced Ns chromosomes belonged to homoeologous group 1. Furthermore, diagnostic fragments of random amplified polymorphic DNA marker OPAG10986 and simple sequence repeat marker Xgwm601 135 were cloned, sequenced, and converted into SCAR markers, i.e., RHS153 and SHS10, respectively, which were validated using a range of distinct plant species and a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22 II). The results demonstrated that the SCAR markers targeted the Ns genome of P. huashanica and they were linked to the 1Ns chromosome. In addition, 12-3 was evaluated to test its leaf rust resistance in the adult stages and its agronomic traits. These newly developed EST–STS and SCAR markers will be powerful tools for wheat breeders who want to screen for genotypes containing the 1Ns chromosome, with low costs and high throughput.

Ploidy manipulation and introgression breeding in Darwin hybrid tulips

Meiotic polyploidisation via crossing with 2n gamete producing genotypes and interploidy crosses are two of the main methods currently used to obtain polyploid tulips. In our study diploid 2n gamete producing F-1 hybrids of Darwin hybrids (Tulipa gesneriana x Tulipa fosteriana) and triploid hybrid resulting from 'Rhodos' x 'Princeps' cross were used as pollen donor and crossed with cultivars of T. gesneriana in the following combination: 2x x 2x, 3x x 2x, 2x x 3x, and 3x x 3x. The progenies resulting from crosses at diploid level were mostly diploid, whereas a few seedlings were triploid. In 3x x 2x crosses aneuploids with chromosome constitution in between triploid and tetraploid (43-45 chromosomes) were predominant, but also one tetraploid (2n = 4x = 48) and four pentaploids (2n = 5x = 60) were obtained. In 2x x 3x crosses most progenies were triploid with the exception of a few aneuploids (3x + 1 and 3x - 1), whereas in 3x x 3x cross diploid and aneuploid genotypes were recorded with chromosome number varied from 27 to 34. These results indicate that triploid parents produced aneuploid as well as euploid (x, 2x, 3x) gametes and that success in ploidy manipulation in tulip depends to a large degree on the ploidy level of the parental genotypes used for hybridization. Genome constitution of selected population of F1 and BC1 hybrids was analyzed through genomic in situ hybridization (GISH). GISH analysis of the BC1 showed a considerable amount of intergenomic recombination which is desirable for introgression breeding.

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids

Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis. Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.

Molecular identification of a new wheat-Thinopyrum intermediumssp.trichophorumaddition line for resistance to stripe rust

Stripe rust, caused by Puccinia striiformis f. sp. tritici, was one of the most disaster foliar diseases for wheat-growing areas of the world. Thinopyrum intermedium has provided novel resistance genes to multi-fungal disease, and new wheat-Th. intermedium derivatives for stripe rust resistance still need to develop for wheat breeding. Wheat line X484-3 was selected from a cross between wheat line MY11 and wheat-Th. intermedium ssp. trichophorum partial amphiploid TE-1508, and was characterized by genomic in situ hybridization (GISH) and functional molecular markers. Chromosome counting revealed that the X484-3 was 2n = 44 and GISH analysis using Pseudoroegneria spicata genomic DNAas a probe demonstrated that X484-3 contained a pair of St-chromosomes from Th. intermedium donor parents. The functional molecular markers confirmed that introduced St-chromosomes belonging to linkage group 7, indicating that line X484-3 was a 7St addition line. The resistance observation displayed that the introduced Th. intermedium ssp. trichophorum derived chromosomes 7St were responsible for the stripe rust resistances at adult plant. The identified wheat-Th. intermedium chromosome 7St addition line X484-3 can be used as a donor in wheat breeding for stripe rust resistance.

Cytogenetic evidence of mixed disomic and polysomic inheritance in an allotetraploid (AABB) Musa genotype

Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widely used. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in its fertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploid genotype. The potential implications of interspecific recombination and chromosomal composition of diploid gametes for Musa improvement are presented. The pairing of different chromosome sets at diploid and tetraploid levels was investigated through a combination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromosomes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was also conducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploid genotype. Analysis of chromosome associations at both ploidy levels suggested that the newly formed allotetraploid behaves as a 'segmental allotetraploid' with three chromosome sets in a tetrasomic pattern, three sets in a likely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploid gametes were detected in progenies, with the chromosome constitution appearing to be more homogenous in pollen than in ovules. Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural AB varieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosome exchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer of valuable alleles.

Genomic in situ hybridization reveals both auto- and allopolyploid origins of different North and Central American hexaploid potato (Solanum sect. Petota) species

Wild potato ( Solanum L. sect. Petota Dumort.) species contain diploids (2n = 2x = 24) to hexaploids (2n = 6x = 72). J.G. Hawkes classified all hexaploid Mexican species in series Demissa Bukasov and, according to a classic five-genome hypothesis of M. Matsubayashi in 1991, all members of series Demissa are allopolyploids. We investigated the genome composition of members of Hawkes's series Demissa with genomic in situ hybridization (GISH), using labeled DNA of their putative progenitors having diploid AA, BB, or PP genome species or with DNA of tetraploid species having AABB or AAA(a)A(a) genomes. GISH analyses support S. hougasii Correll as an allopolyploid with one AA component genome and another BB component genome. Our results also indicate that the third genome of S. hougasii is more closely related to P or a P genome-related species. Solanum demissum Lindl., in contrast, has all three chromosome sets related to the basic A genome, similar to the GISH results of polyploid species of series Acaulia Juz. Our results support a more recent taxonomic division of the Mexican hexaploid species into two groups: the allopolyploid Iopetala group containing S. hougasii, and an autopolyploid Acaulia group containing S. demissum with South American species S. acaule Bitter and S. albicans (Ochoa) Ochoa.

Size and location of radish chromosome regions carrying the fertility restorer Rfk1 gene in spring turnip rape

In spring turnip rape (Brassica rapa L. spp. oleifera), the most promising F1 hybrid system would be the Ogu-INRA CMS/Rf system. A Kosena fertility restorer gene Rfk1, homolog of the Ogura restorer gene Rfo, was successfully transferred from oilseed rape into turnip rape and that restored the fertility in female lines carrying Ogura cms. The trait was, however, unstable in subsequent generations. The physical localization of the radish chromosomal region carrying the Rfk1 gene was investigated using genomic in situ hybridization (GISH) and bacterial artificial chromosome–fluorescence in situ hybridization (BAC–FISH) methods. The metaphase chromosomes were hybridized using radish DNA as the genomic probe and BAC64 probe, which is linked with Rfo gene. Both probes showed a signal in the chromosome spreads of the restorer line 4021–2 Rfk of turnip rape but not in the negative control line 4021B. The GISH analyses clearly showed that the turnip rape restorer plants were either monosomic (2n = 2x = 20 + 1R) or disomic (2n = 2x = 20 + 2R) addition lines with one or two copies of a single alien chromosome region originating from radish. In the BAC–FISH analysis, double dot signals were detected in subterminal parts of the radish chromosome arms showing that the fertility restorer gene Rfk1 was located in this additional radish chromosome. Detected disomic addition lines were found to be unstable for turnip rape hybrid production. Using the BAC–FISH analysis, weak signals were sometimes visible in two chromosomes of turnip rape and a homologous region of Rfk1 in chromosome 9 of the B. rapa A genome was verified with BLAST analysis. In the future, this homologous area in A genome could be substituted with radish chromosome area carrying the Rfk1 gene.

Assessment of intergenomic recombination through GISH analysis of F1, BC1 and BC2 progenies of Tulipa gesneriana and T. fosteriana

Using 23 F1 hybrids, 14 BC1 and 32 BC2 progenies, the genome composition of Darwin hybrid tulips was analysed through genomic in situ hybridisation (GISH) of somatic chromosomes. All plants were diploids (2n = 2x = 24) with the exception of one tetraploid BC1 (2n = 4x = 48) and one aneuploid BC2 (2n = 2x + 1 = 25) hybrid. Morphometric analysis in F1 hybrids revealed a difference in the total length of chromosomes representing genomes of T. gesneriana and T. fosteriana, where the percentage of each genome equaled 55.18 ± 0.8 and 44.92 ± 0.6% respectively. GISH distinguished chromosomes from both parent genomes although there was a lack of consistent chromosome labelling in some cases. In both T. gesneriana and T. fosteriana chromosomes some segments of heterochromatin in the telomeric and intercalary regions exhibited a higher intensity of fluorescence. In situ hybridisation with 5S rDNA and 45S rDNA probes to metaphase chromosomes of F1 hybrids showed that these regions are rich in rDNA. A notable feature was that, despite genome differences, there was a considerable amount of intergenomic recombination between the parental chromosomes of the two species as estimated in both BC1 and BC2 offspring. The number of recombinant chromosomes ranged from 3 to 8 in BC1 and from 1 to 7 in BC2 progenies. All recombinant chromosomes possessed mostly a single recombinant segment derived from either a single crossover event or in a few cases double crossover events. This explains the fact that, unlike the situation in most F1 hybrids of other plant species, certain genotypes of Darwin hybrid tulips behave like normal diploid plants producing haploid gametes and give rise to mostly diploid sporophytes.

Molecular Cytogenetic Characterization of Wheat–Thinopyrum elongatum Addition, Substitution and Translocation Lines with a Novel Source of Resistance to Wheat Fusarium Head Blight

Thinopyrum elongatum (2n=2x=14, EE), a wild relative of wheat, has been suggested as a potentially novel source of resistance to several major wheat diseases including Fusarium Head Blight (FHB). In this study, a series of wheat (cv. Chinese Spring, CS) substitution and ditelosomic lines, including Th. elongatum additions, were assessed for Type II resistance to FHB. Results indicated that the lines containing chromosome 7E of Th. elongatum gave a high level of resistance to FHB, wherein the infection did not spread beyond the inoculated floret. Furthermore, it was determined that the novel resistance gene(s) of 7E was located on the short-arm (7ES) based on sharp difference in FHB resistance between the two 7E ditelosomic lines for each arm. On the other hand, Th. elongatum chromosomes 5E and 6E likely contain gene(s) for susceptibility to FHB because the disease spreads rapidly within the inoculated spikes of these lines. Genomic in situ hybridization (GISH) analysis revealed that the alien chromosomes in the addition and substitution lines were intact, and the lines did not contain discernible genomic aberrations. GISH and multicolor-GISH analyses were further performed on three translocation lines that also showed high levels of resistance to FHB. Lines TA3499 and TA3695 were shown to contain one pair of wheat-Th. elongatum translocated chromosomes involving fragments of 7D plus a segment of the 7E, while line TA3493 was found to contain one pair of wheat-Th. elongatum translocated chromosomes involving the D- and A-genome chromosomes of wheat. Thus, this study has established that the short-arm of chromosome 7E of Th. elongatum harbors gene(s) highly resistant to the spreading of FHB, and chromatin of 7E introgressed into wheat chromosomes largely retained the resistance, implicating the feasibility of using these lines as novel material for breeding FHB-resistant wheat cultivars.

Identification of genome constitutions in Begonia × chungii and its putative parents, B. longifolia and B. palmata, by genomic in situ hybridization (GISH)

Based mainly on morphological features and geographical distribution, Begonia×chungii (2n=22) was recently reported as a natural hybrid between B. longifolia and B. palmata in Taiwan. This study aims to confirm the hybridity of B.×chungii and to sort out the genome constitutions of its putative parents, using genomic in situ hybridization (GISH). Genomic DNAs of both parental species were used as probes for B.×chungii and the experimental F1 hybrid, B. palmata×B. longifolia, in GISH analyses. Bicolor-GISH analyses in B.×chungii showed that the 22 chromosomes consisted of six chromosomes hybridized with a probe derived from the B. palmata genome, six with another probe from the B. longifolia genome and the remaining ten with both genomes overlapped. Meanwhile, bicolor-GISH in B. palmata×B. longifolia showed a remarkable similarity to that of B.×chungii. The reciprocal GISH results between B. longifolia and B. palmata were comparable. Our GISH analyses confirmed that B.×chungii is a natural F1 hybrid between B. longifolia and B. palmata. Genomes of the parental species were shown to be partially homologous, suggesting a derived common ancestral genome in them.

Karyotype and genomic in situ hybridization pattern in ×Brassicoraphanus, an intergeneric hybrid between Brassica campestris ssp. pekinensis and Raphanus sativus

The karyotype and genomic in situ hybridization (GISH) of an intergeneric hybrid Baemoochae, ×Brassicoraphanus, which originated from hybridization between Chinese cabbage, Brassica campestris (synonym, rapa) ssp. pekinensis, and radish, Raphanus sativus, were analyzed to determine its chromosome complement. In the karyotype analysis, B. campestris was verified to have 2n = 20 chromosomes, including a particular pair of the subtelomeric chromosomes with the nucleolar organizer; R. sativus to have 2n = 18 chromosomes, including a particular pair of the submetacentric chromosomes with the secondary constriction of nucleolar organizer; and ×Brassicoraphanus to have 2n = 38 chromosomes, including both the subtelomeric chromosomes of Brassica and the secondary constriction chromosome pair of Raphanus. These findings indicate that ×Brassicoraphanus is a polyploid between Brassica and Raphanus. In the GISH analysis using chromosomes of B. campestris and R. sativus as the probe and blocking DNA, respectively, only 20 chromosomes of Brassica had hybridization signals. This result reveals that ×Brassicoraphanus is an intergeneric hybrid consisting of the complete genomes of both Brassica and Raphanus. However, the nucleolar organizers of Brassica and Raphanus were not identified because the hybridization signals appeared to be centering mainly around the centromere, becoming weak at the edges.