胡萝卜与川西獐牙菜不对称体细胞杂种的核/质基因组特征

胡萝卜(Daucus carota var.sativa)原生质体与经260μW/cm2紫外线照射的川西獐牙菜(Swertia mussotii)原生质体用PEG法诱导融合.对融合再生的克隆的5SrDNA间隔序列和RAPD分析结果得知,各再生克隆均存在双亲的核DNA及重组DNA.杂种的核基因组组成以胡萝卜(受体)为主,供体川西獐牙菜DNA谱带较少;UV照射剂量对体细胞杂种核基因组组成没有明显的影响.进一步对再生克隆叶绿体DNA的SSR分析表明,杂种细胞中双亲叶绿体基因组随机分离并发生重组.     

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。     

山羊草属S基因组与小麦属B／G基因组RAPD标记和特异DNA片段克隆及研究

本研究利用132个随机引物,对山羊草属和小麦属11个种的DNA进行扩增。对其中特异RAPD扩增产物进行克隆,然后用其做探针与小麦族23个种属DNA的RAPD扩增产物进行Southern杂交。共得到24个特异克隆：其中小麦族共有特异克隆1个,山羊草属和小麦属共有特异克隆2个,S基因组特异克隆2类7个,B／G基因组特异克隆2类6个,S基因组与B／G基因组共有的特异克隆8个。24个特异克隆中有22个测定了序列,其中15个为Fasta数据库里显示未见报导的序列。用这24个特异DNA克隆制成的探针与相应的23个材料经HindⅢ酶切消化的总DNA进行Southern杂交,发现其中7个可做为基因组特异探针。通过对24个特异DNA克隆分析研究表明：①S基因组是由两个基本不同的类型构成的,即拟斯卑尔脱山羊草为一个类型,其余4个S组山羊草为另一类型;因此建议前基因型符号仍保留为“S”,而其余4个种之间无明显不同,故应把基因组符号统一为“S^1”②S基因组是小麦B／G基因组的供体,而拟斯卑尔脱山羊草可能是最主要的供体,但并不排除其余S基因组的种参与了B／G基因组形成的可能;研究还表明B基因组与S基因组还是有很大区别的,并已找到了B基因组的特异标记。     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(Triticum aestivum L.)与高冰草(Agropyron elongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8 F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析.结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代.RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定.     

柑橘基因组原位杂交(GISH)技术体系的建立

通过改进实验方法,建立起柑橘基因组原位杂交(Genomic in situ hybridization,GISH)分析技术,并成功地应用于属间有性杂种鉴定,为进一步分析柑橘体细胞杂种核基因组组成奠定了基础。柑橘的染色体制片以去壁低渗-火焰干燥法较好,容易获得大量清晰、分散的有丝分裂中期相,且杂质较少。切刻平移法生物素(Biotin)标记探针,标记基因组总DNA与封阻基因组总DNA的浓度比例为1：50时,能有效分开属间杂种枳橙中来自双亲的染色体。     

普通小麦与钩刺山羊草杂交F1的农艺性状及细胞遗传学的观察

为有效地利用钩刺山羊草（Ａｅｇｉｌｏｐｓ ｔｒｉｕｎｃｉａｌｉｓ Ｌ．）的抗白粉病基因对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）进行遗传改良,了解两者杂交后杂种Ｆ１的遗传机制是十分必要的。对Ｆ１杂种的研究表明,二价体频率高于理论值,是分别存在于钩刺山羊草Ｃ和Ｕ基因组中的小麦５Ｂ染色体上Ｐｈ基因抑制因子联合作用的结果。以尾状山羊草（Ａｅｇｉｌｏｐｓ ｃａｕｄａｔａ Ｌ．）Ｃ基因组特异重复序列     

消减杂交技术结合限制性显示技术制备K562细胞特异基因探针

建立一种简便、快速、特异的制备基因芯片探针的方法.以K562细胞和正常人淋巴细胞作为消减对象,利用自行建立的消减方法进行消减杂交,结合限制性显示技术,分组扩增差异cDNA,回收K562细胞特异基因片段,制作基因芯片探针.结果显示,分离到400个K562特异的基因,片段大小均一,适于制作cDNA芯片.消减杂交技术结合限制性显示技术制备基因芯片探针,具有快速、简便、特异的特点,降低了芯片制作成本,可加速芯片的推广应用.     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(TriticumaestivumL.)与高冰草(Agropyronelongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析。结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代。RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定。     

尾状山羊草与硬粒小麦,普通小麦的杂交及外源染色质检测

尾状山羊草（AegilopscaudataL．）具有丰富的抗病虫和高赖氨酸、高蛋白优良性状,是进行小麦（TriticumaestivumL．）遗传改良的重要遗传资源。合成了硬粒小麦（TriticumdurumDesf．）-尾状山羊草双二倍体、进行了普通小麦与双二倍体的杂交。并以作者克隆的尾状山羊草C基因组特异重复序列PAeca212为探针,对上述杂交后代的花粉母细胞进行了染色体原位杂交检测。证实了新合成的双二倍体中有7对C基因组染色体;在F2中检测到C基因组染色体的自发纯合易位,显示出从C基因组向小麦转移外源基因的光明前景。     

Construction of a primary RH panel of Italian ryegrass genome via UV-induced protoplast fusion

Symmetric and asymmetric somatic hybrids were produced via protoplast fusion between common wheat ( TRITICUM AESTIVUM L.) cv. "Jinan 177" and Italian ryegrass ( LOLIUM MULTIFLORUM Lam.). The ryegrass without or with UV irradiation was used as a donor, providing a small amount of chromatin. In these somatic hybrids, most ryegrass chromosomes have been confirmed preferential elimination and the somatic hybrid calli and plants showed wheat-like morphology. Some of the hybrid lines were used for the analysis of distribution and heredity of donor DNA in the hybrid genome and the possibility of establishing a radiation hybrid (RH) panel of the ryegrass in the present experiment. These hybrids, subcultured for two and three years, retained the ryegrass DNA examined by RFLP and GISH analysis, respectively. Distribution of the ryegrass DNA in the wheat genomes of 20 single-cell individuals, randomly selected from hybrid cell lines produced, were analyzed by 21 ryegrass genome specific SSR markers. The average frequencies of molecular marker retention in symmetric hybrid lines (UV 0), as well as asymmetric hybrid lines from UV 30 s and 1 min were 10.88, 15.48 and 33.86, respectively. It was suggested that the UV dose increased the introgression of donor DNA into wheat genome. The ryegrass SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2 approximately 3 years. This revealed that those asymmetric somatic hybrids are suitable for the introgression of ryegrass DNA into wheat, and for RH panel and RH mapping.     

Different rates of chromosome elimination in symmetric and asymmetric somatic hybridization between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis>

The protoplasts of tall fescue (Festuca arundinacea Schreb.) were fused with those of Bupleurum scorzonerifolium Willd. The latter were irradiated with UV at an intensity of 380 μW/cm² for 0 s (combination I), 30 s (combination II), and 60 s (combination III) before fusion. Putative hybrid calli, leaves, and shoots were generated from the fusion products. They were recognized as somatic hybrids by a combined analysis of chromosome numbers, isozyme, RAPD, and 5S rDNA spacer sequence. The hybrid calli with morphogenetic ability and leaves/shoots differentiation had the B. scorzonerifolium phenotype, whether they were derived from symmetric fusion (UV 0 s) or asymmetric fusion (UV 30 s/60 s). Cytological tests revealed that these hybrids contained the complete set (12) of B. scorzonerifolium chromosomes and 0–4 partner tall fescue chromosomes. The tall fescue chromosomes were rapidly eliminated in combinations II and III, but gradually lost in combination I. It was noted that the green leaves and shoots were produced earlier, and the differentiation frequency was higher in combinations II and III than in combination I, which corresponded to the speed of elimination of the tall fescue chromosomes in the hybrids. Therefore, UV irradiation can indirectly promote elimination of tall fescue chromosomes and hybrid differentiation. B. scorzonerifolium can repel partner chromosomes with mechanism that differs from UV.     

普通小麦与簇毛麦原生质体的紫外线融合

从来源于普通小麦品种济南177（Triticum aestivum cv.Jinan 177）悬浮细胞系的原生质体与来源于簇毛麦（Haynaldia villosa）胚性愈伤组织的原生质体融合获得体细胞杂种。供体簇毛麦原生质体在融合之前用紫外线照射30s或1min,紫外线剂量为360Цw/cm^2。仅由紫外线照射30s的组合获得再生愈伤组织克隆。细胞学、生物化学及PCR分析结果证实了再生克隆的杂种性质。用线粒体基因特异的探针进行的RFLP分析的结果表明,杂种中含有融合双亲的线粒体并且发生了重组。由杂种愈伤组织再生得到白化苗。讨论了紫外线对融合产物的影响。     

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.     

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。     

Intermediate fertile Triticum aestivum （＋）Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.     

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant

Morphology, the extent of elimination of donor chromosomes and the organelle composition of highly asymmetric somatic hybrid plants between a interspecific tomato hybrid Lycopersicon esculentum x L. pennellii (EP) as donor and a Solarium melongena, eggplant (E), recipient, were studied. Morphologically, the somatic hybrids most resemble eggplant but, due to polyploidy, growth is slower relative to both fusion parents. The somatic hybrids produce flowers that are characterized by abnormal styles, stigmas and by anthers which do not produce pollen. Limited amounts of donor EP genomic DNA were found in the three somatic hybrid plants (H18-1, H18-2 and H18-3), by dot-blot hybridization with probe pTHG2, equivalent to 6.23,5.41, and 5.95% EP, respectively. These percentages translated to the presence of 3.59, 2.90 and 3.19 average-size EP chromosomes in plants H1 8-1,-2 and-3, respectively. RFLP determination of L. esculentum- and L. pennellii-specific chromosomes revealed that only fragments of eight to ten out of the 24 EP chromosomes (EP has 12 L. esculentum and 12 L. pennellii chromosomes) are present in the asymmetric somatic hybrid plants. Loci of L. esculentum and L. pennellii were evenly represented in plants H18-1, -2, and -3: four to five from L. esculentum and four to five from L. pennellii. All somatic hybrid plants retained locus TG22, chromosome 4, from both EP species. Although the regeneration of plants, H18-1, -2 and-3 was from one callus, loci TG31 and TG79 of L. esculentum chromosome 2 and L. pennellii chromosome 9, respectively, were missing in hybrid plant H18-1. The three somatic hybrid plants all had chloroplast DNA fragments specific for S. melongena. The mitochondrial genome (mtDNA) in the asymmetric somatic hybrids showed predominantly the pattern of eggplant; however, some eggplant-specific polymorphic bands were not present in the three plants.     

Molecular analysis of the extent of asymmetry in two asymmetric somatic hybrids of tomato

Summary Two somatic hybrid plants generated from a single fusion event between Lycopersicon esculentum and irradiated L. pennellii protoplasts have been analyzed at the molecular level. Over 30 loci have been analyzed using isozymes and RFLPs. All loci tested on chromosomes 2–10 were heterozygous, while those loci on chromosome 12 were homozygous L. pennellii in both somatic hybrids. In one of the somatic hybrids, 2850, loci on chromosome 1 were also homozygous L. pennellii. The other somatic hybrid, 28F5, was heterozygous at all chromosome 1 loci tested, but exhibited altered stoichiometry of parental bands as compared to the sexual hybrid. Loci on chromosome 2 from both somatic hybrids have altered stoichiometry, with L. pennellii alleles being four times more abundant than expected. Both somatic hybrids contain the L. esculentum chloroplast genome, while only L. pennellii polymorphisms have been detected in the mitochondrial genome.     

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.