Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis

Electrofusion of mesophyll protoplasts from two male sterile dihaploid Solanum tuberosum genotypes. DHAK-11 and DHAK-33, was performed. Selection of putative fusion products was based on vigorous callus growth. Regeneration of rooted putative hybrid plants was scored 14 weeks after fusion. Characterization of hybrids was performed by use of morphological assessment, random amplified polymorphic DNA (RAPD), and cytological and isozyme analysis. The rate of regenerated hybrids from callus was ca 6%. Of the putative hybrids, 45% were confirmed as true hybrids. Morphological assessment of the putative hybrids revealed that tetraploid and neartetraploid hybrids were vigorous plants with intermediate characteristics between the two parental phenotypes in respect to internode length, leaf size and shape, and purple pigmentation on the abaxial side of the leaves. Near-hexaploid hybrids were slender plants with small leaves and short petioles. Selected RAPD primers showed unique marker bands for the two parental genotypes. Hybrid plants revealed the unique marker bands from both parents. A total of 53 randomly chosen decamer primers were tested and 26 primers (49%) detected polymorphism between the two dihaploid parentals. Two primers revealed that one parental marker band was missing in two aneuploid hybrids. However, of 51 putative hybrids, a double test with two independently chosen primers showed unequivocally the hybrid character of 23 plants. The ploidy level of the hybrids was analysed by chromosome numbers in root tip cells and by number of guard cell chloroplasts. A strong correlation between the chromosome number and the number of chloroplasts was obtained. The hybrid nature of all RAPD-verified hybrids was confirmed by isozyme analysis with malate dehydrogenase.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

Behaviour of mouse primary spermatocyte nuclei after fusion to enucleated metaphase II oocytes

Primary spermatocytes originating from prepubertal mouse testes were electrofused to metaphase II (MII)-stage oocytes, enucleated either by the conventional micromanipulation method or by chemical treatment with etoposide and cycloheximide. These experiments were followed by assessment of morphological changes in transferred nuclei using light microscopy, by chromosomal analyses and by screening of hybrids for the presence or absence of DNA synthesis using anti-bromodeoxyuridine antibody and immunofluorescence staining of the hybrids. The results show differences between the two types of ooplasts in susceptibility to activation stimuli. However, when activated, both types of ooplasts gave rise to hybrids of similar morphology. From 35.3% to 63% of activated hybrids originating from chemically or microsurgically enucleated oocytes, respectively, contained one large pronucleus in cytoplasm, 62% or 31.6% hybrids from those two groups, respectively, possessed two smaller pronuclei and a few contained three or four pronuclei. No DNA synthesis was detected in any hybrid containing one or more pronuclei. The chromosome spreads of hybrids with premature chromosome condensation (PCC) morphology (before activation) show that most of the hybrids had a diploid (2n) number of chromosomes. The nature and regularity of the cell division cycle in the hybrids are discussed.     

Genetic transfer of a twospotted spider mite (Acari: Tetranychidae) repellent in tomato hybrids

Lycopersicon hirsutum Dunal is very resistant to arthropod herbivory, and research on causes of resistance has often implicated trichomes and their secretions. To better understand relationships among resistance, repellency, and 2,3-dihydrofarnesoic acid, a trichome-borne sesquiterpenoid spider mite repellent, two tomato, Lycopersicon esculentum Miller, varieties were interbred with a highly resistant, spider mite repellent accession (LA1363) of L. hirsutum. Backcross and F2 generations were produced with each tomato variety. Whole leaves of 99 hybrids were bioassayed with twospotted spider mites, Tetranychus urticae Koch, allowing selection of six hybrids (two susceptible and four resistant) for each generation of each family. When these 24 hybrids were characterized for spider mite repellency with thumbtack bioassays, two hybrids had repellent leaflets, demonstrating that repellency was genetically transferred to interspecific tomato hybrids. Leaflet washes containing trichome secretions from each of three hybrids, including the two having repellent leaflets, were repellent in bridge bioassays. For the two hybrids having repellent leaflets and leaflet washes, removal of trichome secretions by dipping leaflets in methanol eliminated leaflet repellency. 2,3-Dihydrofarnesoic acid was present in trichome secretions of the hybrids having leaflet repellency, and it also was present in secretions of other hybrids, indicating that its presence is essential, but not sufficient for leaflet repellency. With regard to resistance, 16 of the hybrids tested had been identified as resistant in a whole leaf bioassay, but only two had repellent leaflets, indicating that other mechanisms of resistance are present in the resistant L. hirsutum parent.     

三种普通欧洲鲤科鱼类(拟鲤、粗鳞鳊和欧鳊)人工杂交F1代形态学观察

对反交试验获得的18个月的拟鲤、粗鳞鳊和欧鳊F1代进行了鉴定.对不同于拟鲤、粗鳞鳊和欧鳊三种鱼的可量和可数形态特征分析表明,拟鲤×欧鳊和拟鲤×粗鳞鳊杂交种比粗鳞鳊×欧鳊杂交种与它们的双亲更容易区分.拟鲤杂交种间具有很多形态上的相似性,例如,上侧虹膜在拟鲤为红色,杂交种则介于双亲之间.在粗鳞鳊×欧鳊杂交种中也可以观察到居间类型.拟鲤×粗鳞鳊和拟鲤×欧鳊各自的杂交种可以通过眼睛的大小、较少的侧线鳞数和2行咽喉齿等特征相区分.粗鳞鳊×欧鳊杂交种与另外两个杂交种相比,具有更多的臀鳍分支鳍条数和一个中央具一黑线的虹斑.三个种进行杂交再获得的互交种通常不易区分.     

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Summary Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.     

青花菜与萝卜属间杂种的表型和分子鉴定

应用形态学、细胞学和分子标记的方法对通过胚挽救获得的青花菜与萝卜属间杂种植株进行了鉴定。结果表明:杂种植株的生长势明显强于父本和母本,大部分形态性状超出了父、母本的范围,部分性状介于父母本之间或偏向于父本或母本一方。表型和细胞学观察显示杂种植株具有类似母本的雄性不育特性。SSR分子标记分析表明,杂种植株包含了双亲的遗传信息。可见,远缘杂种是双亲间的真杂种。远缘杂交种的获得与初步鉴定为促进萝卜属和芸薹属的基因交流提供了桥梁种质,为进一步创制萝卜或芸薹属作物新种质提供了基础材料。     

The RNA-DNA hybrid structure determined by EPR, CD and RNase H1

A or B: RNA-DNA hybrids, key intermediates in gene regulation, were classified by pulsed electron-electron double resonance (PELDOR) in combination with CD spectroscopy into two classes, interpreted as A- and B-like structures. RNase H1 cleavage of these hybrids is in full agreement with these assignments, cleaving the hybrids with A-like geometry preferentially. This combined analytical approach allows the interpretation and eventually the design of more easily cleavable hybrids as needed for the antisense technology.     

双单杂交技术选育亮盖灵芝与白肉灵芝优良性状杂交子

以亮盖灵芝Ganoderma lucidum和白肉灵芝G. leucocontextum为亲本,采用亮盖灵芝异核体双核菌丝与通过原生质体单核化获得的白肉灵芝单核体进行双单杂交,最终获得34株杂交子。采用ISSR技术对34株杂交子及亲本进行DNA多态性聚类分析,杂交子主要分为两个类群。对杂交子进行生长速度和灵芝酸含量对比,筛选到4株优质杂交子。进一步进行杂交子及亲本间HPLC指纹图谱对比,发现4株杂交子中有3株HPLC指纹图谱在具备两个亲本特征灵芝酸的同时,还有低极性灵芝酸的产生。     

Genome differentiation in Magonoliaceae as revealed from meiotic pairing in interspecific and intergeneric hybrids

The cross compatibility within and between Yulania Spach and Michelia L.(Magnoliaceae) is relatively good and various such hybrids,obtained by conventional artificial hybridization,are available.The aim of the present study was to determine the extent of genome differentiation between the species involved in these crosses through the observation of chromosome pairing during meiosis in pollen mother cells (PMCs) of the hybrids.Chromosome pairing behavior was studied in five species (2n =38) and two interspecific hybrids of Michelia,eight species (2n =38,76 and 114) and 10 interspecific hybrids of Yulania,and three intergeneric hybrids between Michelia and Yulania.The results showed that chromosome pairing was normal with bivalent formation in diploid parental species and in interspecific hybrids.In addition to bivalents,multivalents were encountered in polyploid parental species and polyploid interspecific hybrids.In the intergeneric hybrids between a tetraploid Yulania and two diploid Michelia,19 chromosomes,most likely originating from Michelia,were unable to synapse from zygotene to metaphase I.Meiotic chromosome pairing indicated a high degree of homology between species within Michelia and Yulania and less homology between the genomes of these two genera.The differentiation of morphological characters and the distinctness of natural distribution also support the conclusion that these two genera are likely independent monophyletic groups.This suggests that the two genera were split at early evolution of Magnoliaceae and the overlapping characteristics in external morphology and internal structures of the two genera may be the result of parallel evolution or ancient common ancestry.     

Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination

By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.     

Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.     

HYBRIDS AND PHYLOGENETIC SYSTEMATICS II. THE IMPACT OF HYBRIDS ON CLADISTIC ANALYSIS

I examined three aspects of the cladistic treatment of a set of 17 F₁ hybrids of known parental origin: (1) impact of hybrids on consistency index (CI) and number of most parsimonious trees (Trees), (2) placement of hybrids in cladograms, and (3) impact of hybrids on hypotheses of relationship among species. The hybrids were added singly and in randomly selected sets of two to five to a data set composed of Central American species of Aphelandra (including the parents of all hybrids). Compared to analyses with the same number of OTUs all of which were species, the analyses with hybrids yielded results with significantly higher CI. There was no difference in Trees between analyses with hybrids versus species. There was thus no evidence that hybrids would appear to be more problematic for cladistic methods than species. Accordingly, hybrids will not be readily identifiable as taxa that cause marked change in these indices. About % of the hybrids were placed as the cladistically basal members of the lineage that included the most apomorphic parent. Relatively apomorphic hybrids were placed proximate to the most derived parent (ca. 13% of hybrids). Other placements occurred more rarely. The most frequent placements of hybrids thus did not distinguish them from normal intermediate or apomorphic taxa. When analyses with hybrids yielded multiple most parsimonious trees, these were no more different from each other than were the equally parsimonious trees that resulted from analyses with species. Most analyses with one or two hybrids resulted in minor or no change in topology. When hybrids caused topological change, they frequently caused rearrangements of weakly supported portions of the cladogram that did not include their parents. When they disrupted the cladistic placement of their parents, they often caused their parents to change positions, with at least one topology bringing the parental lineages into closer proximity with the hybrid placed between them. Hybrids between parents from the two main lineages of the group caused total cladistic restructuring. In fact, the degree of relationship between a hybrid's parents (measured by both cladistic and patristic distance) was strongly correlated with CI (negatively) and with the degree of disturbance to cladistic relationships (positively). Thus, hybrids between distantly related parents resulted in cladograms with low CI and major topological changes. This study suggests that hybrids are unlikely to cause breakdown of cladistic structure unless they are between distantly related parents. However, these results also indicate that cladistics may not be specially useful in distinguishing hybrids from normal taxa. The applicability of these results to other kinds of hybrids is examined and the likely cladistic treatment of hybrids using other sources of data is discussed.     

Schizophrenia-associated chromosome 11q21 translocation: Identification of flanking markers and development of chromosome 11q fragment hybrids as cloning and mapping resources

Genetic linkage, molecular analysis, and in situ hybridization have identified TYR and D11S388 as markers flanking the chromosome 11 breakpoint in a large pedigree where a balanced translocation, t(1;11)(q43;q21), segregates with schizophrenia and related affective disorders. Somatic cell hybrids, separating the two translocation chromosomes from each other and from the normal homologues, have been produced with the aid of immunomagnetic sorting for chromosome 1– and chromosome 11–encoded cell-surface antigens. The genes for two of these antigens map on either side of the 11q breakpoint. Immunomagnetic bead sorting was also used to isolate two stable X-irradiation hybrids for each cell-surface antigen. Each hybrid carries only chromosome 11 fragments. Translocation and X-irradiation hybrids were analyzed, mainly by PCR, for the presence of 19 chromosome 11 and 4 chromosome 1 markers. Ten newly designed primers are reported. The X-irradiation hybrids were also studied cytogenetically, for human DNA content, by in situ Cot1 DNA hybridization and by painting the Alu-PCR products from these four lines back onto normal human metaphases. The generation of the translocation hybrids and of the chromosome 11q fragment hybrids is a necessary preliminary to determining whether a schizophrenia-predisposition gene SCZD2 is encoded at this site.     

Daphnia Galeata × D. Longispina Hybrids in Western Norway

We describe the occurrence of D. galeata×longispina hybrids in two lakes of western Norway. Parental species and interspecific hybrids were characterised by both nuclear and mitochondrial molecular markers. In one of the populations, hybrids were shown to dominate the population over several years. A few individuals in both populations were probably not F1 hybrids, but possibly backcrosses or F2 hybrids. Most (possibly all) F1 hybrids were of D. galeata maternal origin. In addition, interspecific hybrids could be identified based on morphological characters, which were intermediate between the parental species. Interspecific hybridisation between these two species is remarkable, since they are distantly related.     

Masculinization mechanism of hybrids in bitterlings (Teleostei: Cyprinidae)

The sex ratio of bitterling hybrids (subfamily: Acheilognathinae) is often likely to be biased toward males. Artificial hybridization was carried out in 10 species of bitterlings (three genera) in order to elucidate the masculinization mechanism of hybrids. Tanakia himantegus never produced viable F1 hybrids with other species, while hybrids of most other species were viable. In terms of sex ratio and fertility, hybrids were clearly divided into two groups: congeneric Tanakia hybrids and others. Both male and female congeneric Tanakia hybrids were fertile. The sex ratio was nearly 1:1 in all groups of Tanakia hybrids. Except for the congeneric Tanakia hybrids, sterile males appeared predominantly in groups of hybrids in which females were very rare but remained fertile. Sterile intersexes were also observed in five hybrid groups: T. lanceolata (female) x Acheilognathus cyanostigma (male), Rhodeus uyekii (female) x T. lanceolata (male), A. rhombeus (female) x T. lanceolata (male), A. rhombeus (female) x T. limbata (male), and A. tabira tabira (female) x A. cyanostigma (male). In the development of male-predominant hybrids, although hybrid and control (parental species) hatching and survival rates do not differ, no females appeared in hybrids, contrary to the controls. Taking the female heterogametic sex-determining system (ZW) and the phylogenetic relationship of bitterlings into consideration, the masculinization mechanism of hybrids in bitterlings can be explained by the interaction of two sex chromosomes, derived from each parental species. The basic genetic sex in bitterlings is male (ZZ) and the derivative is female (ZW). When parental species are related, the sex phenotype of hybrids coincides with the genetic sex. However, when the parental species differ, the sex phenotype of the ZW genotype is reversed to become male by an abnormal interaction between the Z and W chromosomes. The rare appearance of females and intersexes in male-predominant hybrids might be due to complete or partial functional expression of the W chromosome.     

Transgenic parthenocarpic eggplants: superior germplasm for increased winter production

Winter production of three eggplant hybrids transgenic for the parthenocarpic gene DefH9-iaaM was compared, in an unheated greenhouse, to the performance of two untransformed control hybrids and the commercial parthenocarpic cultivar Talina. Each hybrid was either treated or untreated with a commercial formulation of phytohormones to induce fruit set and growth. The productivity of the transgenic parthenocarpic hybrids was not influenced by the hormonal treatment. On the contrary, the productivity of untransformed hybrids was significantly improved by hormonal treatment of the flower buds. The yield of the transgenic hybrids was significantly higher than that obtained in the corresponding untransformed hybrids, even when the latter were treated with phytohormones. The yield increment due to the parthenocarpic trait was particularly evident when compared to the yield of the two corresponding hybrid combinations, which are identical except for the presence of the DefH9-iaaM gene. The transgenic hybrids allowed an increase in productivity of ca. 25%. This increment coincided with a 10% reduction in cultivation cost, mainly due to the labour needed for the hormonal sprays, and to the production of fruits of better quality. Thus, the DefH9-iaaM gene is a biotechnological tool superior to both agronomic and traditional genetic parthenocarpic mutants.     

Mitochondrial DNA patterns are similar in gametosomatic and somatic hybrids of two Nicotiana species

Summary The segregation and recombination patterns of mitochondrial genome in the somatic hybrids of Nicotiana tabacum and N. rustica were studied by RFLP analysis using four heterologous mitochondrial DNA probes, namely cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), 26s rDNA and 5s-18s rDNA. These RFLP patterns were compared with those of the gametosomatic hybrids of these two species. A preponderance of N. rustica type patterns was observed in the somatic hybrids. One of the somatic hybrids had N. rustica type pattern with COI probe, novel pattern with COII, and 26s rDNA probe and N. tabacum type pattern with 5s-18s rDNA probe. These patterns are identical to those of some of the gametosomatic hybrids and could only be due to the recombination of mitochondrial genomes of the two parents. The extent and the nature of recombination of mitochondrial genomes is similar in gametosomatic and somatic hybrids.