Regulation of nuclear DNA synthesis in amoeba interspecific hybrids

Interspecific hybrids between A. proteus and A. indica which have different durations of nuclear S periods have been produced by reciprocal nuclear transfer after enucleating the host cells. The duration of nuclear DNA synthesis was studied in the clones of these interspecific hybrids and parental stocks by ³H-thymidine autoradiography. These studies showed that nuclear S period of the hybrids changed to that characteristic to the nuclear component of the parental cell from which the hybrid's original cytoplasm was derived. The results of these studies were interpreted as evidence for cytoplasmic regulation of the rate of chromosome replication.     

DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrid/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that at the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis.     

Species-specific repetitive DNA used to identify interspecific somatic hybrids

Plasmid DNA clones containing repetitive DNA sequences were isolated from Hyoscyamus muticus and Nicotiana tabacum. Non cross-hybridizing probes from each species were used in a simple hybridization test with DNA isolated from presumptive somatic hybrids. This allowed unequivocal identification of DNA from both species in the hybrids.     

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia

Restriction fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F. angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.     

DNA methylation in placentas of interspecies mouse hybrids

Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.     

姜花属种间杂交胚拯救研究

为提高姜花属种间杂交胚挽救中幼胚萌发率,以白姜花×金姜花的胚珠为试材,研究不同胚珠发育时期、不同培养基及低温处理果实对幼胚萌发率的影响.结果表明,白姜花×金姜花胚挽救的适宜培养基是MS+0.1 mg/L BA十0.1 mg/L NAA;接种时期以60 d的幼胚培养效果最佳;低温处理果实3～6 d能有效提高幼胚的萌发率.     

Inheritance of freezing resistance in interspecific F1 hybrids of Eucalyptus

Summary The inheritance of freezing resistance in interspecific F₁ hybrid families of Eucalyptus encompassing 27 different species combinations and a range of levels of hardiness was examined. Freezing resistance was assessed by determining the temperatures required to cause either 30% (T30), 40% (T40), or 50% (T50) leakage of electrolytes from excised leaf discs subjected to artificial freezing. Highly significant variation in freezing resistance occurred between species; the maximum difference between parents in any specific combination was over 9°C (E. gunnii x E. globulus). Freezing resistance was inherited in a predominantly additive manner in interspecific hybrids, although there was a tendency towards partial dominance toward the more sensitive species in some combinations (e.g., E. nitens x E. Globulus, E. nitens x E. camaldulensis, E. gunnii x E. globulus). The full expression of this genetic variation appeared to increase with hardiness and in some cases appeared to vary with ontogeny. Estimates of individual narrow-sense heritability of freezing resistance for pure E. nitens families were h ² = 0.66±0.44 and 0.46±0.44. Across all species combinations examined, the heritability of F₁ family means estimated from midparent regression was h ² = 0.76±0.06 and h ² = 0.89±0.06 for T40 and T50 values, respectively. The advantage of using selected parents for interspecific hybridization is demonstrated and the implications of these results for breeding for freezing resistance in Eucalyptus are discussed.     

Analysis of DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrids/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that of the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 195–205. The text was submitted by the authors in English.     

水曲柳下胚轴的组织培养和植株再生

1　植物名称　水曲柳 (Fraxinusmandshurica)。2　材料类别　成熟种子的下胚轴。3　培养条件　诱导培养基 :MS +TDZ 0 .3mg·L- 1 (单位下同 ) +IBA 0 .1 ;伸长培养基 :MS + 6 BA 1 .0 +IBA 0 .5 ;增殖培养基 :MS + 6 BA 2 .0 +IBA 0 .0 5 ;生根培养基 :MS +IBA 0 .5。所有培养基均加 2 %蔗糖、0 .5 %琼脂 ,pH调至 5 .8,1 2 1℃高温、高压灭菌 2 0min。培养温度为 ( 2 5± 1 )℃ ,湿度为 60 %～ 70 % ,光照度为 1 5 0 0～ 2 0 0 0lx ,光照时间为 1 6h·d- 1 。4　生长与分化情况4.1　无菌材料的获得　秋季选取水曲柳饱满种子 ,…     

水曲柳种子次生休眠的预防和解除

以解除休眠和经高温诱导产生次生休眠的水曲柳种子为材料,探讨预防和解除水曲柳种子次生休眠的方法。结果表明：GA3和乙烯利不能阻止已解除休眠水曲柳种子在25℃下萌发时诱导的次生休眠。干燥和短时间低温解除水曲柳种子次生休眠的效果不明显,较长时间（大于2周）的低温对解除水曲柳种子的次生休眠有一定的效果。综合来看,10^-3mol·L^-1的乙烯利或GA4＋7解除水曲柳种子次生休眠的效果较好。     

Production and characterization of interspecific hybrids between Brassica maurorum and crop brassicas

 Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum×B. napus, B. maurorum×B. carinata, B. maurorum×B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques. Received: 31 July 1998 / Accepted: 14 August 1998     

水曲柳FmWRKY44基因克隆及表达分析

克隆水曲柳FmWRKY44基因,探究其在非生物胁迫和激素胁迫中的作用。利用水曲柳干旱转录组序列设计特异引物,克隆FmWRKY44基因的完整ORF序列,并对该序列及其编码产物进行生物信息学分析,采用qRT-PCR技术分析FmWRKY44表达模式。克隆了一个水曲柳WKRY基因,编码区长1383bp,编码460氨基酸。对其编码蛋白分析发现其为稳定亲水蛋白,亚细胞定位预测主要在细胞核,进行保守域及同源性分析分析,属于WRKYⅠ类家族,命名为FmWRKY44。qRT-PCR分析发现,FmWRKY44在种子中高度表达,并不同程度响应低温、高温、盐和干旱4种非生物胁迫。同时发现FmWRKY44与NAA、ABA、GA3、JA植物激素响应,水曲柳FmWRKY44基因积极响应低温、高温胁迫。     

水曲柳腋芽离体快繁研究初报

以水曲柳带顶芽、腋芽茎段为外植体进行离体培养,研究其适宜的灭菌方法、基本培养基种类和激素对腋芽萌发、丛芽产生、芽苗增殖的影响。结果表明,水曲柳的腋芽茎段为快繁的适宜外植体,茎段灭菌以用0.05%HgCl₂处理2 min最好。在萌芽培养中,BA和2ip均可促进腋芽萌发,但以8 mg·L^-1 BA处理时萌发效果最好,萌发率达100％;将腋芽萌发后长成的新枝转入添加ZT的培养基中,出现丛芽,在添加1.0 mg·L^-1的ZT的培养基中增殖效果最好,增殖系数达到3.0。无论在萌芽培养还是增殖培养中均发现WPM培养基最适合水曲柳腋芽的离体快繁。     

Hydraulic properties of individual xylem vessels of Fraxinus americana

Studies of the hydraulic properties of xylem vessels have been limited to measurements of whole plant or whole stem segments. This approach allows the longitudinal transport properties of the ensemble of vessels within a stem to be determined, but provides little information on radial transport. Here the xylem of Fraxinus americana L. has been examined using a new method that allows the transport properties of individual vessels to be examined. One goal of this study was to quantify transport parameters relevant to embolism repair. The longitudinal conductivity of vessel segments open at both ends (i.e. no end walls) agreed with values predicted by the Poiseuille equation. Radial specific conductance (conductance per unit area) was approximately six orders of magnitude lower than the longitudinal conductance of the vessel segment normalized by the cross-sectional area of the vessel lumen. There was a step increase in the radial specific conductance of previously gas-filled vessels when the delivery pressure exceeded 0.4 MPa. This is consistent with the idea that positive pressure, required for embolism repair, can be compartmentalized within a vessel if the bordered pit chambers are gas-filled. The diffusion coefficient for the movement of gas from a pressurized air-filled vessel was of the same order of magnitude as that for air diffusing through water (1.95 e(-9) m(2) s(-1)). Estimates of the time needed to displace all of the gas from an air-filled vessel were in the order of 20 min, suggesting that gas removal may not be a major limitation in embolism repair.     

Plastid DNA inheritance and plastome-genome incompatibility in interspecific hybrids of Zantedeschia (Araceae)

Plastid DNA (ptDNA) probes were used in RFLP analysis to determine ptDNA inheritance in interspecific hybrids in Zantedeschia. Biparental and maternal ptDNA inheritance was found in albino hybrids between the evergreen species Z. aethiopica and several winter-dormant species. From two albino hybrids, different types of ptDNA were detected in shoots derived from different parts of an embryo. This result indicates that plastids were sorted out during embryo development. Only maternal ptDNA was detected in the hybrids of Z. aethiopica × Z. odorata (a summer-dormant species) but paternal, biparental, and maternal ptDNA were found in the hybrids of the reciprocal cross. Z. odorata × Z. aethiopica. By correlating these ptDNA inheritance patterns with the leaf colour (albino, pale-green, and green) of the hybrids, it is suggested that the Z. odorata plastome is incompatible with the Z. aethiopica genome. The Z. aethiopica plastome is partially compatible with the Z. odorata genome but the development of Z. aethiopica plastids appears to be blocked by the presence of the Z. odorata plastids.     

Allele-specific DNA methylation analyses associated with siRNAs in Arabidopsis hybrids

Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.