RFLP analysis and genomic in situ hybridization (GISH) in somatic hybrids and their progeny between Lycopersicon esculentum and Solanum lycopersicoides

 RFLP (restriction fragment length polymorphism) and GISH (genomic in situ hybridization) analyses were employed to identify the chloroplast and nuclear genomes of the somatic hybrids and progeny between tomato ‘Ohgata zuiko’ and Solanum lycopersicoides (‘LA 2386’). A random distribution of the chloroplast genotype was determined using a cloned 19.6-kb BamHI fragment (Ba1) of tobacco chloroplast DNA. Eight selected hybrids were analyzed for their chromosomal compositions; 4 were tetraploids (2n=48) with an equal number of chromosomes derived from each parent as accurately determined by GISH, and the other 4 were hexaploids, containing an average of two sets of tomato chromosomes and one set from the wild parent. RFLP analysis with six tomato nuclear probes of known chromosomal locations revealed no major variation among the 44 hybrid plants surveyed. However, it also showed the presence of both parent-specific alleles and the loss of some and the presence of a few non-parental alleles, indicating rearrangement and/or recombination of the nuclear DNA. The relevance of the molecular and cytological methods and the potential use of somatic hybrids for plant breeding are demonstrated. Received: 20 July 1997 / Accepted: 6 October 1997     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Regeneration and molecular characterization of intergeneric somatic hybrids between Citrus reticulata and Poncirus trifoliata

Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC₃) and selfed (S₃) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC₃ plants (originated from somatic hybrids 3, 4, 10) with 2–8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

Summary Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (Trovita orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.     

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.     

甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。     

甜橙与酸橙体细胞杂种核质组成鉴定

采用流式细胞术(flow cytometry,FCM)、简单重复序列(simple sequence repeat,SSR)和酶切扩增多型性序列(cleaved amplifiedpolymorphic sequence,CAPS)等技术分析酸橙(Citrus aurantium L.)叶肉原生质体和甜橙(C.sinenis Osbeck cv.Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种.FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体.用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本.在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种.通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样.结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本.讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用.     

Transmission of organelle genomes in citrus somatic hybrids

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interspecific hybridization and the analysis of meiotic chromosome pairing inDahlia (Asteraceae — Heliantheae) species with x = 16

The successful production of a large number of artificial hybrids betweenDahlia species based on x = 16 has allowed a detailed study of their genomic relationships. Chromosome behaviour in these artificial hybrids was extremely similar to that observed in parental species suggesting that there is a considerable degree of homology between the genomes of theseDahlia species. Using GISH it can be demonstrated that in these hybrids bivalent formation involved pairing only between parental genomes. The ability of GISH to differentiate between parental genomes in artificial hybrids was variable, indicating that molecular divergence of highly repeated sequences has accompanied the evolution of these species. However, the extent of chromosome pairing and chiasma formation in the hybrids does not reflect the differences that can be detected by GISH. Seyeral of the new hybrid combinations have resulted in horticulturally interesting plants.     

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root-tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.     

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.     

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.     

Analysis of remote asymmetric somatic hybrids between common wheat and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Callus-derived protoplasts of common wheat (Triticum aestivum L. cv. Hesheng 3) irradiated with ultraviolet light were fused by using the PEG method with cell suspension-derived protoplasts of Arabidopsis thaliana. Regenerated calli and green plants resembling that of wheat were obtained. The hybrid nature of putative calli and plants were confirmed by isozyme, random amplified polymorphic DNA and genomic in situ hybridization (GISH) analyses. GISH results indicated that 1∼3 small chromosome fragments of A. thaliana were found introgression into the terminals of wheat chromosomes, forming highly asymmetric hybrids. Cytoplasmic genome tests did not show any cytoplasmic genetic materials from A. thaliana. However, variations from the normal wheat cytoplasmic genome were found, indicating recombination or rearrangement occurred during the process of somatic hybridization. The chromosome elimination in the asymmetric somatic hybridization of remote phylogenetic relationship was discussed. A miniature inverted-repeat transposable element related sequence was found by chance in the hybrids which might accompany and impact the process of somatic hybridization. Jingyao Deng and Haifeng Cui provided same contribution to this work.     

Rapid alterations of DNA sequence and cytosine methylation induced by somatic hybridization between Brassica oleracea L. var. italica and Brassica nigra (L.) Koch

Somatic hybrids were produced between hypocotyl protoplasts of Brassica oleracea L. var. italica (broccoli) and mesophyll protoplasts of B. nigra (black mustard) using polyethylene glycol—mediated protoplast fusion. A total of fifteen somatic hybrids derived from six calli (no. 1, 3, 8, 21, 38 and 44) were obtained. Cytological analysis showed that all the hybrids possessed 2n = 34, the sum of the parental chromosomes and the genomic in situ hybridization analysis revealed their BBCC genome constitutes. Moreover, all the hybrids exhibited different type of meiosis abnormalities, which were more usually observed in pollen mother cells at metaphase II/anaphase II (MII/AII, 16.1–39.6 %) than at metaphase I/anaphase I (MI/AI, 7.8–15.2 %). Simple sequence repeat analysis revealed that all the hybrids showed the same cytoplasmic genome as broccoli. Structure and methylation-variation of the nuclear were investigated by amplified fragment length polymorphism (AFLP) and DNA methylation-sensitive amplification polymorphism (MSAP). Our results indicated that all the hybrids mainly had the AFLP and MSAP banding patterns from the addition of two parents plus some alterations. The incidences of the AFLP polymorphic bands in the hybrids showed a range of 9.8–18.7 % while the DNA methylation alteration in the hybrid no. 38 was 4.07 %. This result suggested that somatic hybridization could induce more DNA sequence changes than methylation alterations in the early stage of allotetraploid hybrids.     

GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus

New Brassica napus inbred lines with different petal colors and with canola quality and increased levels of oleic (∼70%, 10% higher than that of B. napus parent) and linoleic (28%) acids have been developed in the progenies of one B. napus cv. Oro × Orychophragmus violaceus F₅ hybrid plant (2n=31). Their genetic constituents were analyzed by using the methods of genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). No intact chromosomes of O. violaceus origin were detected by GISH in their somatic cells of ovaries and root tips (2n=38) and pollen mother cells (PMCs) with normal chromosome pairing (19 bivalents) and segregation (19:19), though signals of variable sizes and intensities were located mainly at terminal and centromeric parts of some mitotic chromosomes and meiotic bivalents at diakinesis or chromosomes in anaphase I groups and one large patch of chromatin was intensively labeled and separated spatially in some telophase I nuclei and metaphase II PMCs. AFLP analysis revealed that substantial genomic changes have occurred in these lines and O. violaceus–specific bands, deleted bands in ‘Oro’ and novel bands for two parents were detected. The possible mechanisms for these results were discussed.