True Hybrids Research Articles

Red Rice (Oryza sativa) Plant Types Affect Growth, Coloration, and Flowering Characteristics of First- and Second-Generation Crosses with Rice

Red rice is a major weed of rice in the southern U.S. and can intercross with rice. Knowledge of the plant phenotypes from such crosses would be valuable for identification and management of these plants. Male-sterile long-grain tropical japonicas ‘Kaybonnet-1789’ and ‘Cypress-1819’ were crossed with two awned and two awnless U.S. red rice types. F1 plants produced pubescent leaves, red pericarp, and medium-grain seeds. Crosses involving awned LA3 and TX4 red rice produced F1 plants with reddish-purple basal leaf sheaths and usually flowered within the same time periods as the parents, whereas those involving awnless StgS red rice had green basal leaf sheaths, flowered much later than either parent, and produced awnless F1 and F2 offspring. Crosses involving awned red rice produced F1 plants with long awns and F2 plants with awns ranging in length from zero to that of red rice parents. F1 plants were taller than either parent and produced intermediate culm angles similar to red rice, whereas F2 plants had culms ranging from erect (like rice) to more open than red rice. Thus, true F1 hybrids from crosses between pure breeding (homozygous) rice and red rice can be positively identified by a combination of traits including pubescent leaves, medium-grain seeds with red pericarps, open plant types, and heights greater than the red rice parent. F1 hybrids may be awned or awnless, have purple or green stems, or have normal or delayed heading. F2 plants have a broad combination of phenotypic traits found in both parents and F1 hybrids.

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.

BREEDING STRATEGIES TO INCREASE GENETIC VARIABILITY WITHIN HIBISCUS SYRIACUS

In an attempt to introgress an increased growth vigour and improved flowering into H. syriacus two different breeding strategies were followed. A first approach focused on creating hexaploid H. syriacus plants. This was done by crossing tetraploid H. syriacus cultivars with colchicine doubled (octoploid) H. syriacus `Oiseau Bleu' seedlings. The generated hexaploid progeny had an increased growth vigour compared to tetraploid cultivars. The F1 seedlings were also sterile. Out of the hexaploid F1 population an improved blue flowered plant was selected and multiplied. The second strategy emphasized on the interspecific hybridization between H. syriacus and H. paramutabilis. When used as a seed parent, H. paramutabilis failed to set fruits. However, in reciprocal crosses flowers reacted by fruit setting. Also in these crosses a high number of fruits aborted in an early stage of development. Germination of the surviving seeds occurred within 2-3 weeks. The generated seedlings grew vigorously. Leaves of all seedlings had an intermediate morphology compared to those of H. paramutabilis and H. syriacus, indicating that the obtained progenies were true interspecific hybrids.

Generation of cell hybrids via a fusogenic cell line

Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult. We have established a stable cell line expressing a viral fusogenic membrane glycoprotein (FMG) that is not itself susceptible to fusion. This cell line has been used to generate hybrids and to evaluate the relevance of tools used for hybrid detection. This FMG-expressing cell line promotes fusion between autologous or allogeneic TC and DC in any combination, generating 'tri-parental hybrids'. At least 20% of TC are found to be integrated into hybrids. It is speculated that this tri-parental hybrid approach offers new possibilities to further modulate the anti-tumour effect of the DC/TC hybrids since it allows the expression of relevant immunostimulatory molecules by appropriate engineering of the fusogenic cell line.

Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers

Abstract Although there have been some attempts to obtain intergeneric hybrids between Japanese pear (Pyrus pyrifolia Nakai) and apple (Malus × domestica Borkh.) mature hybrids have not been generated due to hybrid lethality. Shoots from immature hybrid embryos were gamma irradiated and cultured at normal temperature conditions to obtain viable intergeneric plants. Gamma irradiation doses of 50 and 100 Gy cultured at 25 °C killed all shoots but there was one survivor at 150 Gy and 25 °C. Hybrid status of the shoot was confirmed by using morphology, flow cytometry and SSR markers. The intergeneric plant had similar leaf serrations to apple and similar petioles to Japanese pear. Flow cytometric analysis revealed that the intergeneric shoot had a DNA content intermediate between the parents. SSR analysis showed one allele from each parent for all 16 SSR loci investigated. These data strongly suggest that the obtained shoot is a true hybrid between Japanese pear and apple.

Utility of a fertility restorer gene linked marker for testing genetic purity of hybrid seeds in rice (Oryza sativa L.)

2 Topic: Genetics Key Author: A.K. Singh Correspondence Address: Division of Genetics Indian Agricultural Research Institute New Delhi 110 012 India E-mail: aks_gene@mailcity.com Authors: A. Garg, A.K. Singh, K.V. Prabhu, T. Mohapatra, N.K. Tyagi, N. Nandakumar, R. Singh and F.U. Zaman Utility of a fertility restorer gene linked marker for testing genetic purity of hybrid seeds in rice (Oryza sativa L.) Pages: 9-18 The utility of a fertility restorer (R f ) gene linked co-dominant STMS marker RM258 was examined for testing the genetic purity of hybrid seeds in rice. DNA isolated from 450 seedlings of a superfine grain aromatic rice hybrid Pusa RH10, was PCR amplified using RM258. Of these, 15 plants were found homozygous for either of the parental type (CMS line Pusa 6A and restorer line PRR 78) allele. The rest 435 plants were heterozygous at the RM258 locus indicating their true hybrid nature. The genetic purity of hybrid seed lot was thus found to be 96.67%. The results of molecular analysis were confirmed by the Grow Out Test (GOT). Further, the efficacy of RM258 was compared with markers, RM201, 206, 216, 228, 247 and 263, which were not linked to R f gene but were polymorphic between the corresponding A and R lines. Some of the plants identified as hybrid, using RM258 appeared as self of the parental lines at certain unlinked marker loci. Similarly, among the 15 plants homozygous for either of the parental type alleles at the RM 258 locus, the distribution of homozygous and heterozygous genotypes at unlinked marker loci was observed in varying frequency. These observations indicated that the assessment of genetic purity of hybrid seeds based on unlinked markers might not be fully reliable. The study suggests that a single R f gene linked co-dominant marker can provide a precise and quick alternative to GOT, for testing hybrid seed purity.

Interspecific hybridization in vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers

Vanilla, Vanilla planifolia Andrews, is native to Mexico and Central America, but is now cultivated in other parts of the tropics. Continuous clonal propagation has resulted in very little variability for crop improvement programmes in vanilla. In this study, an attempt has been made to increase the spectrum of variation by interspecific hybridization with Vanilla aphylla, an Indian species which is tolerant to Fusarium. Interspecific hybrids were successfully produced and morphological characters and molecular profiles revealed the true hybridity of the progenies. Ten seedling progenies of V. planifolia, and four interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) using a number of different loci as markers were evaluated and 319 amplified fragment length polymorphisms (AFLPs) and 83 random amplified polymorphic DNAs (RAPDs) loci were marked. The profiles indicate similarity between the parents, selfed progenies and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla. This is the first report in vanilla, indicating that RAPD and AFLP profiles coupled with morphological characters can be utilized to assess the variability and hybrid nature of genotypes and of successful interspecific hybridization and production of hybrids between V. planifolia and V. aphylla.

Species assignment and hybrid identification among Scandinavian hares Lepus europaeus and L. timidus

In Scandinavia, suspected hybrids between the native mountain hare Lepus timidus and the introduced brown hare L. europaeus have been observed by hunters since the first introductions of brown hares in the late 19th century. Several attempts to verify the status of these suspected hybrids have been unsuccessful. Recently, however, the transmission of mitochondrial DNA (mtDNA) from mountain hares to brown hares was documented and interpreted as a consequence of hybridisation and subsequent introgression. However, mtDNA markers alone will not allow identification of first-generation hybrids because of the strictly maternal inheritance of mitochondria. Here, we combine mtDNA data with analyses of variation in seven microsatellite loci among brown hares, mountain hares and putative hybrids. Our purpose was to determine species differentiation in nuclear DNA markers, elucidate the extent of interspecific gene flow, identify true hybrids within our sample and evaluate the ability of hunters to identify hybrids. The estimated genetic difference between species was low (FST = 0.18 – 0.24, RhoST = 0.09 – 0.16). We believe these low estimates result from a reticulated mode of evolution among hares, with repeated gene flow over the species barrier. Population assignment tests and randomly assembled, artificial, hybrid genotypes were used to classify individuals independently from the morphologically assessed species identity. More than half (57%) of the putative hybrid specimens were assigned unambiguously to either species and first-generation hybrids seem to be rare. Morphological plasticity and backcrossing, which confound species identification within the genus Lepus, might explain invalid classifications by hunters.

Characterization of Intergeneric Hybrids of Saccharum using Molecular Markers

The potential use of RAPD markers in identifying genuine intergeneric hybrids of Saccharum was examined in 12 hybrids of Saccharum with Erianthus spp., Sorghum bicolor and Zea mays. In the three groups of hybrids examined, RAPD markers could precisely identify the true hybrids of Saccharum officinarum × Sorghum bicolor and Saccharum officinarum × Zea mays. Though Erianthus had a very distinct RAPD profile and as many as 107 Erianthus-specific bands were identified, a decisive profile that can identify all the hybrids could not be obtained. The strong molecular differentiation between S.officinarum and Erianthus was not adequately resolved in the RAPD profiles of their hybrid progenies which suggest that more refined molecular techniques will be required for the characterization of Saccharum–Erianthus hybrids.

Overspill routing in optical networks: a true hybrid optical network design

To efficiently support the highly dynamic traffic patterns of the current Internet in large-scale switches, we propose a new hybrid optical network design: Overspill Routing In Optical Networks (ORION). By taking advantage of the reduced (electronic) processing requirements of all-optical wavelength switching, the electronic bottleneck is relieved. At the same time, ORION achieves a level of statistical multiplexing comparable to the more traditional point to point WDM solutions, circumventing the bandwidth inefficiencies of all-optical wavelength switched networks, caused by dynamic traffic patterns. The result is a true hybrid optical network design, forming a bridge between these two switching concepts. In this paper the generic concept of ORION is described. An example node design, based on current advanced optical technologies, is described in detail. The ORION concept is also evaluated, comparing it with its two composing technologies, optical wavelength switching and point to point WDM, as well as a third, more trivial, hybrid one, through several case studies

Variation in Crossability among Rhododendron japonicum f. flavum Plants as Pollen Parents when Crossed with Evergreen Species, R. eriocarpum

An attempt was made to obtain the paternal parental stock to hybridize evergreen azaleas (seed parent) with Rhododendron japonicum (A. Gray) J. V. Suringar f. flavum Nakai (pollen parent). To examine the variation in hybridization barriers and crossability, pollen from nine accessions of R. japonicum f. flavum were used to pollinate one plant of R. eriocarpum (Hayata) Nakai. Significant differences among R. japonicum f. flavum accessions appeared in fertilization rate, mature seed numbers per capsule, albino rate, survival rate, and the crossability. Pollination with pollen of R. japonicum f. flavum accessions 27026136 and 27026139 yielded a relatively large number of viable seedlings per pollinated flower as compared to the others. Our results suggest that it is important to select highly crossable R. japonicum f. flavum plants as the pollen parent to obtain true hybrids.

Introgression from wild Cicer reticulatum to cultivated chickpea for productivity and disease resistance

AbstractInterspecific hybridization is known to improve productivity and resistance to diseases in many crops. Therefore, an attempt was made to introgress productivity and disease resistance into chickpea from wild Cicer species. The true F1 hybrids of cultivated chickpea genotypes ‘L550’ and ‘FGK45’ with C. reticulatum were backcrossed twice to their cultivated female parents to minimize the linkage drag of undesirable wild traits. The pedigree method was followed to advance the segregating populations from straight crosses (without backcross) and BC1/BC2 generations to F5–F7. The interspecific derivatives recorded up to a 16.9% increase over the check cultivars and a 25.2% increase over the female parent in a preliminary yield evaluation trial. Of the 22 interspecific derivatives thus derived, four desi and two kabuli lines were further evaluated for seed yield in replicated trials at three diverse locations. These lines possess a high degree of resistance to wilt, foot rot and root rot diseases, and recorded a 6.1–17.0% seed yield increase over the best check cultivars.

The great tit (Parus major) - a misclassified ring species

The great tit (Parus major) has been considered to be the most typical example of an avian ring species. The terminal taxa of the ring (major and minor sectors) are supposed to be reproductively isolated in a zone of secondary contact in the middle Amur valley, Siberia. Our study combines molecular markers (cytochrome-b), bioacoustic analyses and morphological characters to judge the ring species status of the great tit complex. Despite a notable percentage of intermediately coloured birds in the mixed population of middle Amur, a lack of mitochondrial introgression between the major and minor sectors and a small number of true hybrids among voucher specimens from this area suggest at least a partial reproductive barrier between both sectors. In contrast, variation of morphological and especially acoustic characters along the ring-shaped area and the phylogenetic structure of the P. major group do not match the ring species concept. Bioacoustic and molecular data (cytochrome-b sequences) reveal two large and closely related subspecies blocks, the sectors major and bokharensis in the Western Palaearctic and central Asia, and the sectors minor and cinereus in the Eastern Palaearctic and South-east Asia, respectively. The two western sectors diverged only recently (0.5 Mya) and they were separated from the eastern group by Pleistocene events about 1.5 Mya. Songs from allopatric regions of the two subspecies blocks differ distinctly in frequency parameters and element composition. In the area of secondary contact, males of all phenotypes share the same frequency range of song, close to the range of the typical minor song. Hybrids and major males sing mixed repertoires of typical major and minor strophe types as well as mixed strophes. In contrast, phenotypic minor males display only pure minor strophes. Differences in mate choice and mating success based on repertoire size are believed to uphold the reproductive barrier between major and minor birds in the area of sympatry. Taxonomic consequences suggest three separate species in the Parus major complex: Parus major s.s. (including the very closely related bokharensis sector), Parus minor and Parus cinereus. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 153–174.

Induced Androgenesis in Fish: Obtaining Viable Nucleocytoplasmic Hybrids

A self-review of long-standing studies of induced androgenesis in fish. The data are provided on the induction of diploid androgenesis in teleost fish via blocking the first cleavage division and the development of the method of dispermic androgenesis in sturgeon fish. Androgenetic nucleocytoplasmic hybrids were obtained using the method of dispermic androgenesis and eggs of previously produced true interspecific hybrids. Specific morphological features of androgenetic hybrids were described and dispermic androgenesis was realized using cryopreserved sperm. The mechanism of sex determination in sturgeon fish is discussed with reference to possible us of androgenesis for restoration of rare and disappearing species.

The Use of DNA Sequences from the β-Amylase Gene to Determine the Genetic Relationship among Sweetpotato, I. batatas and Other Ipomoea Species in Series Batatas (Convolvulaceae)

The sweet potato Ipomoea batatas (L.) Lam. is classified in series Batatas (Choisy) in Convolvulaceae, with 12 other species and an interspecific true hybrid. The phylogenetic relationships of a sweetpotato cultivar and 13 accessions of Ipomoeas in the series Batatas were investigated using the nucleotide sequence variation of the nuclear-encoded β-amylase gene. First, flowers were examined to identify the species, and DNA flow cytometry used to determine their ploidy. The sweetpotato accession was confirmed as a hexaploid, I. tabascana a tetraploid, and all other species were diploids. A 1.1–1.3 kb fragment of the β-amylase gene spanning two exons separated by a long intron was PCR-amplified, cloned, and sequenced. Exon sequences were highly conserved, while the intron yielded large sequence differences. Intron analysis grouped species currently recognized as A and B genome types into separate clades. This grouping supported the prior classification of all the species, with one exception. The species I. tiliacea was previously classified as a B genome species, but this DNA study classifies it as an A genome species. From the intron alignment, sequences specific to both A and B genome species were identified. Exon sequences indicated that I. ramosissima and I. umbraticola were quite different from other A genome species. Placement of I. littoralis was questionable: its introns were similar to other B genome species, but exons were quite different. Exon evolution indicated the B genome species evolved faster than A genome species. Both intron and exon results indicated the B genome species most closely related to sweetpotato (I. batatas) were I. trifida and I. tabascana.

Synthesis and biological activity of mustard derivatives of combretastatins

A series of chimeric compounds bearing the combretastatin and the nitrogen mustard cores were synthesized. All the compounds were cytotoxic and inhibited tubulin polymerization. When combretastatin was joined to chlorambucil via an ester linkage, the resultant compound proved to be significantly more potent than the two compounds put together. When combretastatin was joined to nitrogen mustard via an ether linkage or when a true hybrid was synthesized, loss of potency was observed. Nonetheless, these latter compounds appeared to be more efficacious and surprisingly were able to inhibit tubulin depolymerization at high concentrations.

Molecular criteria for determining new hybrid species—An application to the Sonneratia hybrids

The possible hybrid origin of new species can usually be corroborated by molecular means. Here, we suggest that the segregation patterns of the molecular markers be further analyzed. A true hybrid species should show the patterns under continuous breeding among its members, at least beyond the F2 generation. We applied the guidelines to the putative hybrid species of Sonneratia, a widespread mangrove genus, and concluded that all the observed hybrids in this genus are simple F1’s. Thus, S. × gulngai and S. × hainanensis are not true hybrid species. The segregation patterns of molecular markers should be heeded in interpreting the existence of hybrid species.

Application of ISSR, RAPD, and cytological markers to the certification of Picea mariana, P. glauca, and P. engelmannii trees, and their putative hybrids

Picea glauca (white spruce) and P. engelmannii (Engelmann spruce) are so similar and integrated that it is impossible to distinguish between them and their hybrids using morphological characteristics. Although natural hybrids between P. glauca and P. mariana (black spruce) do not generally occur, even though the 2 species are sympatric in North America, a first-generation hybrid, called the Rosendahl spruce, has been reported in the literature. In this study, several inter-simple sequence repeat (ISSR) markers were developed, as were randomly amplified polymorphic DNA (RAPD) markers, to certify spruce trees and their hybrids. ISSR fingerprinting was more efficient than RAPD assay; it detected 70% polymorphic DNA markers among the spruce species analyzed, whereas RAPD fingerprinting detected only 53%. Species-diagnostic ISSR and RAPD markers differentiating P. glauca from P. engelmannii and P. mariana were cloned and sequenced. Molecular certification of the spruce samples analyzed confirmed that all the seeds from interior spruce populations were true hybrids of P. glauca and P. engelmannii. But the analysis of seeds derived from the putative Rosendahl spruce indicated that this tree is likely a pure P. glauca genotype, rather than a hybrid of P. glauca and P. mariana. These data were confirmed by cytological analyses. Further analysis, using a more sensitive DNA amplification method with designed primers flanking the species-diagnostic ISSR and RAPD markers, revealed that such sequences are not generally species-specific because they are present in other spruce species.

Chromosome numbers of 13 taxa and 12 crossing combinations in Magnoliaceae

Chromosome numbers of 13 taxa and 12 crossing combinations in the Magnoliaceae are reported. The counts of 12 taxa are reported for the first time. The intergeneric crossing combination between Magnolia×soulangeana “Hongyuanbao” (♀), a tetraploid with 2n=4x=76, and Michelia yunnanensis (♂), a diploid with 2n=2x=38, had the number of 2n=3x=57, a half of the total chromosome number of its parents. Similarly, the intergeneric crossing combination between Magnolia×soulangeana “Hongyuanbao” (♀) and Michelia foveolata (♂), a diploid with 2n=2x=38, also had the number of 2n=3x=57. These indicate that the offspring of the two crossing combinations are true hybrids.

Size and ligand effects on the electrochemical and spectroelectrochemical responses of CdSe nanocrystals

The electrochemical properties of CdSe quantum dots with electrochemically inactive surface ligands (TOPO) have been investigated in comparison with the analogous nanocrystals containing electrochemically active oligoaniline ligands. The TOPO-capped nanocrystals have been studied in a wide size range (from 3 to 6.5 nm) with the goal to amplify the influence of the quantum confinement effect on the electrochemical response. The determined HOMO and LUMO levels have been found in good agreement with the ones obtained from photoluminescence studies and those predicted theoretically. Ligand exchange with aniline tetramer significantly influences the voltammetric peaks associated with the HOMO oxidation and the LUMO reduction of the quantum dots, which are shifted to higher and lower potentials, respectively. These shifts are interpreted in terms of the positive ligand charging which precedes the oxidation of the nanocrystals and the insulating nature of the ligand in the case of the nanocrystal reduction. The ligand-nanocrystal interactions have also been studied by UV-Vis-NIR and Raman spectroelectrochemistry in comparison with a specially prepared model compound which, apart from the anchoring function is identical to the grafted oligoaniline ligand. Both spectroelectrochemical techniques clearly indicate the same nature of the oxidation/reduction pathway for both the model compound and the grafted ligand. The influence of the grafting is manifested by a shift in the onset of the ligand oxidation as compared to the case of the "free" model compound. Since both components (ligands and nanocrystals) mutually influence their electrochemical and spectroelectrochemical properties, the newly developed system can be considered as a true molecular hybrid. Such hybrids are of interest because the potential zone of the ligand electroactivity is well separated from that of the nanocrystals and, as a result, the organic part can be electrochemically switched between the semiconducting and the conducting states with no change in the oxidation state of the nanocrystal. The newly developed system offers therefore the possibility of an electrical addressing of individual nanocrystals via the conducting ligands.