Genetic characterization of asymmetric somatic hybrids between Bupleurum scorzonerifolium Willd and Triticum aestivum L.: potential application to the study of the wheat genome

In this paper, we describe how Bupleurum scorzonerifolium/Triticum aestivum asymmetric somatic hybrids can be exploited to study the wheat genome. Protoplasts of B. scorzonerifolium Willd were irradiated with ultraviolet light (UV) and fused with protoplasts of common wheat (T. aestivum L.). All cell clones were similar in appearance to those of B. scorzonerifolium, while the regenerated plantlets were either intermediate or B. scorzonerifolium-like. Genotypic screening using isozymes showed that 39.3% of cell clones formed were hybrid. Some of the hybrid cell clones grew vigorously, and differentiated green leaves, shoots or plantlets. DNA marker analysis of the hybrids demonstrated that wheat DNA was integrated into the nuclear genomes of B. scorzonerifolium and in situ karyotyping cells revealed that a few wheat chromosome fragments had been introgressed into B. scorzonerifolium. The average wheat SSR retention frequency of the RH panel was 20.50%, but was only 6.67% in fusions with a non-irradiated donor. B. scorzonerifolium chromosomes and wheat SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2.5–3.5 years. We suggest the UV-induced asymmetric somatic hybrids between B. scorzonerifolium Willd and T. aestivum L. have the potential for use in the construction of an RH map of the wheat genome.     

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ～501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

混合小麦亲本与新麦草不对称体细胞杂交体系的建立

以小麦品种济南177悬浮细胞系来源的原生质体与同品系胚性愈伤组织制备的原生质体混合后作为受体;以经过380μＷ/ｃｍ²紫外线照射1min、2min的新麦草原生质体分别作为供体,用PEG法诱导融合。组合Ⅰ(176+cha9+新麦草UV 1min)获得16个再生克隆。经过形态学、同工酶、染色体和RAPD分析,确定其全部为属间体细胞杂种。其中的5个克隆再生杂种植株。用7对小麦SSR引物对杂种克隆的叶绿体基因组进行了分析;组合Ⅱ(176+cha9+新麦草UV 2min)只获得3个克隆,且逐渐褐化死亡。表明以小麦济南177的两种培养细胞混合作受体的融合体系有利于杂种的获得及再生;紫外线对融合产物的生长发育有明显的剂量效应。     

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.     

Progress of chromosome engineering mediated by asymmetric somatic hybridization

Plant somatic hybridization has progressed steadily over the past 35 years. Many hybrid plants have been generated from fusion combinations of different phylogenetic species, some of which have been utilized in crop breeding programs. Among them, asymmetric hybrid, which usually contains a fraction of alien genome, has received more attention because of its importance in crop improvement. However, few studies have dealt with the heredity of the genome of somatic hybrid for a long time, which has limited the progress of this approach. Over recent ten years, along with the development of an effective cytogenetical tool ＂in situ hybridization （ISH）＂, asymmetric fusion of common wheat （Triticum aestivum L.） with different grasses or cereals has been greatly developed. Genetics, genomes, functional genes and agricultt, ral traits of wheat asymmetric hybrids have been subject to systematic investigations using gene cloning, genomic in situ hybridization （GISH） and molecular makers. The future goal is to fully elucidate the functional relationships among improved agronomic traits, the genes and underlying molecular mechanisms, and the genome dynamics of somatic introgression lines. This will accelerate the development of elite germplasms via somatic hybridization and the application of these materials in the molecular improvement of crop plants.     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

Production and characterization of asymmetric hybrids between upland cotton Coker 201 (Gossypium hirsutum) and wild cotton (G. klozschianum Anderss)

Asymmetric somatic hybrids were obtained between Gossypium hirsutum Coker 201 and wild cotton G. klozschianum Anderss. An investigation on the effect of ultraviolet (UV) irradiation on donor protoplasts was carried out, and the lethal dose was determined to be 38.7 J cm⁻². We firstly screened the putative hybrids by the color of the calli produced, followed by morphological, cytological, and molecular analysis of putative hybrid plants. Most regenerated plants derived from fused protoplasts displayed a recipient-like morphology, while some showed an intermediate phenotype between Coker 201 and G. klozschianum. Chromosome numbers in these somatic hybrids ranged from 54 to 74. The hybrids were verified by random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR). Absence or co-existence of parents’ genome DNA fragments was identified through molecular analysis. The heredity of cytoplasm was investigated by cleaved amplified polymorphic sequence (CAPS) analysis using mitochondrial and chloroplast universal primer pairs. The results indicated that recombination and rearrangements might have occurred in some regions of mitochondria (mt) and chloroplast (cp) DNA. To our knowledge, this is the first report about asymmetric protoplast fusion in cotton, and the hybrids obtained would be useful for breeding programs.     

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.     

RAPD method for the identification of intergeneric asymmetric somatic hybrid plants of wheat]

Randomly amplified polymorphic DNA (RAPD) method was used to identify the hybrid nature of three kinds of intergeneric asymmetric somatic hybrid plants of wheat: wheat (Triticum aestivum) + Haynaldia villosa, Wheat + Leymus chinensis and wheat + Agropyron elongatum. It was shown from the electrophoresis profiles that the genome of somatic hybrid plants contained specific section genome of both parents after DNA amplification with arbitrary primers. A specific RAPD product (DNA fragment of 0.77 kbp) of A. elongatum generated with primer OPJ-12 was isolated, purified, labeled and used as a probe. Southern blot from OPJ-12 primer-generated specific section genome of the hybrid (T. aestivum + A. elongatum) hybridized to this probe (0.77 kbp) proved that they are homologous in nature. This paper also discussed the advantage of RAPD method in identification of hybrid plants, especially asymmetric somatic hybrids.     

用RAPD技术鉴定小麦属间不对称体细胞杂种

用随机引物扩增多态DNA(RAPD)技术对三种不同组合:小麦(Triticum aestivum)( )簇毛麦(Haynaldia villosa);小麦( )羊草(Leymus chinensis)和小麦( )高冰草(Agropyron elongatum)的属间不对称杂种进行分子鉴定,不同杂种植株的基因组经随机引物扩增后,均出现双亲的多态特异产物,证实它们含有双亲的基因组。将引物OPJ-12扩增的高冰草多态特异产物(分子量为0.77bp的DNA片段)分离纯化并标记作探针,用Southern杂交证明了小麦( )高冰草杂种经OPJ-12扩增的0.77kbp特异片段与高冰草这一片段具有同源性。本文结果证明,RAPD技术可作为小麦属间不对称体细胞杂种的一种快速、简便、有效的分子鉴定方法。     

Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi

Suspension-derived protoplasts of Agropyron elongatum irradiated by ultra-violet light (UV) were fused with the suspension-derived protoplasts of Triticum astivum using PEG. Fertile intergeneric somatic hybrid plants were produced and various hybrid lines have been selected and propagated in successive generations. Their hybrid nature was confirmed by analysis of profiles of isozymes, RAPDs, and 5S rDNA spacer sequences, and via GISH analysis. By the procedure described, the phenotype and chromosome number of wheat could be maintained besides transfer of a few chromosomes and chromosomal fragments from the donor A. elongatum. The results above indicated that highly asymmetric fertile hybrid plants and hybrid progenies of wheat were produced via somatic hybridization.     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

The behavior of <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm² UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm² for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.     

小麦与多年生黑麦草原生质体的电融合及杂种愈伤组织的形成

多年生黑麦草(Lolium perenne L.)悬浮培养细胞来源的原生质体和小麦(Triticumaestivum L.)含叶绿体的悬浮培养细胞来源的原生质体间,用直流方波脉冲进行电融合,获得了体细胞杂种愈伤组织。小麦的原生质体经过碘乙酰胺失活。愈伤组织的形态和颜色被用作识别预期杂种的标记。为了对杂种愈伤组织进行同工酶分析,观察了亲本的9种同工酶谱,其中3种在亲本间表现出差异(ADH、GOT 和 SDH)。酒精脱氢酶(ADH)的分析结果表明,有6个细胞系表现出杂种带。这些细胞系经过其他两种同工酶分析和 rDNA 探针杂交试验表明,一个细胞系表现出基本完全的亲本基因组间的组合,其余5个细胞系是部分杂种。     

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.     

Molecular-genetic analysis of wheat (T. aestivum L.) genome with introgression of Ae. cylindrica Host genetic elements

Wheat-aegilops hybrid plants Triticum aestivum L. (2n = 42) x Aegilops cylindrica Host (2n = 28) were investigated with using microsatellite markers. In two BC1F9 lines some genome modifications connected with losing DNA fragments of initial variety or appearing of Aegilops genome elements were detected. In some investigated hybrids new amplicons lacking in parental plants were found. Substitution of wheat chromosomes for aegilops chromosomes was not revealed. Analysis of microsatellite loci in BC2F5 plants showed stable introgression of aegilops genetic elements into wheat; elimination of some transferred aegilops DNA fragments in the course of backcrossing; decreasing size of introgressive elements after backcrossing. Introgressive lines were classified according to genome changes.     

Transfer of small chromosome fragments of Agropyron elongatum to wheat chromosome via asymmetric somatic hybridization

~~Transfer of small chromosome fragments of Agropyron elongatum to wheat chromosome via asymmetric somatic hybridization1 .Dong,Y.C,GenePools of common wheat,Journal of Triticeae CroPs(in Chinese),2000,20(3):78-81. 2 .Wei,Y.M.,Zheng,YL.Zhou,R.H., Detectlon of the rye chro- matin in multisPikelet wheat germplasm 10-A background using fluorescence in situ hybridization(FISH)and RFLP markers,Acta Bot.Sinica(in Chinese),1999,41(7):722-725. 3 .Xiang,E N.,Xia,G M.…     

Transfer of small chromosome fragments of<Emphasis Type="Italic">Agropyron elongatum</Emphasis> to wheat chromosome via asymmetric somatic hybridization

The chromosome constitution of hybrids and chromatin patterns of Agropyron elongatum (Host)Neviski in F₅ somatic hybrid lines -1–3 and I-1-9 between Triticum aestivum L. and A. elongatum were analyzed. Based on the statistic data of pollen mother cells, F₅ I-1-9 and-1-3 had 20–21 bivalents with a frequency of 84.66% and 85.28%, of which, 89.83% and 89.57% were ring bivalents. The result indicated that both hybrid lines were basically stable in the chromosome constitution and behavior. RAPD analysis showed that the two hybrids contained biparental and integrated DNA. GISH (Genome in situ hybridization) revealed that in the form of small chromosome segments, A. elongatum chromatin was scattered on 4–6 wheat chromosomes near by the region of centromere and telomere in the two hybrid lines. SSR analysis indicated that A. elongatum DNA segments were distributed on the 2A, 5B, 6B and 2D wheat chromosomes in the hybrids, which was in accordance with the GISH results that small-segments intercalated poly-site.