Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.     

Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe--a landmark approach for Thinopyrum genome research

The introduction of alien genetic variation from the genus Thinopyrum through chromosome engineering into wheat is a valuable and proven technique for wheat improvement. A number of economically important traits have been transferred into wheat as single genes, chromosome arms or entire chromosomes. Successful transfers can be greatly assisted by the precise identification of alien chromatin in the recipient progenies. Chromosome identification and characterization are useful for genetic manipulation and transfer in wheat breeding following chromosome engineering. Genomic in situ hybridization (GISH) using an S genomic DNA probe from the diploid species Pseudoroegneria has proven to be a powerful diagnostic cytogenetic tool for monitoring the transfer of many promising agronomic traits from Thinopyrum. This specific S genomic probe not only allows the direct determination of the chromosome composition in wheat-Thinopyrum hybrids, but also can separate the Th. intermedium chromosomes into the J, J(S) and S genomes. The J(S) genome, which consists of a modified J genome chromosome distinguished by S genomic sequences of Pseudoroegneria near the centromere and telomere, carries many disease and mite resistance genes. Utilization of this S genomic probe leads to a better understanding of genomic affinities between Thinopyrum and wheat, and provides a molecular cytogenetic marker for monitoring the transfer of alien Thinopyrum agronomic traits into wheat recipient lines.     

Dramatic genotypic difference in, and effect of genetic crossing on, tissue culture-induced mobility of retrotransposon Tos17 in rice

Key message

We show for the first time that intraspecific crossing may impact mobility of the prominent endogenous retrotransposon Tos17 under tissue culture conditions in rice.

Abstract

Tos17, an endogenous copia retrotransposon of rice, is transpositionally active in tissue culture. To study whether there exists fundamental genotypic difference in the tissue culture-induced mobility of Tos17, and if so, whether the difference is under genetic and/or epigenetic control, we conducted this investigation. We show that dramatic difference in tissue culture-induced Tos17 mobility exists among different rice pure-line cultivars sharing the same maternal parent: of the three lines studied that harbor Tos17, two showed mobilization of Tos17, which accrued in proportion to subculture duration, while the third line showed total quiescence (immobility) of the element and the fourth line did not contain the element. In reciprocal F1 hybrids between Tos17-mobile and -immobile (or absence) parental lines, immobility was dominant over mobility. In reciprocal F1 hybrids between both Tos17-mobile parental lines, an additive or synergistic effect on mobility of the element was noticed. In both types of reciprocal F1 hybrids, clear difference in the extent of Tos17 mobility was noted between crossing directions. Given that all lines share the same maternal parent, this observation indicates the existence of epigenetic parent-of-origin effect. We conclude that the tissue culture-induced mobility of Tos17 in rice is under complex genetic and epigenetic control, which can be either enhanced or repressed by intraspecific genetic crossing.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

Sequence variation and structural conservation in the D-loop region and flanking genes of mitochondrial DNA from Japanese pond frogs

The nucleotide sequences of the D-loop region and its flanking genes of the mitochondrial DNA (mtDNA) from Japanese pond frogs were determined by the methods of PCR, cloning, and sequencing. The frogs belonged to two species, one subspecies, and one local race. The gene arrangements adjacent to the D-loop region were analyzed. The frogs shared a unique mitochondrial gene order that was found in Rana catesbeiana; i.e., cyt b--D-loop region--tRNA(Leu(CUN))--tRNA(Thr)--tRNA(Pro)--tRNA(Phe)--12S rRNA. The arrangements of the three tRNA genes of these frogs were different from those of X. laevis, a species which has the same overall structure as in mammals. Highly repetitive sequences with repeat units (16-bp or 17-bp sequence specific for each taxon) were found in the D-loop region. The length of repetitive sequences varied from 0.6 kbp to 1.2 kbp, and caused the extensive size variation in mtDNA. Several short sequence elements such as putative TAS, OH, CSB-1, and CSB-2 were found in the D-loop region of these frogs. The sequences of these short regulatory elements were conserved in R. catesbeiana, X. laevis, and also in human. The comparison of sequence divergences of the D-loop region and its adjacent genes among various taxa revealed that the rates of nucleotide substitutions depend on genes. The nucleotide sequences of the 3'-side segment of the D-loop region were the most variable among taxa, whereas those of the tRNA and 12S rRNA genes were the most conservative.     

水稻剑叶突变体的表型及T-DNA旁邻基因分析

从已构建的水稻（Oryza sativa L.）T-DNA插入突变体中鉴定获得一株穗部额外发育出叶片的突变体,并根据该叶片的形态学位置将其命名为剑叶突变体（J4）。研究表明这种额外发育的叶片呈现明显的缺陷,主要表现为叶片短小、表皮细胞变小、叶片中维管束数目减少等。进一步通过TAIL-PCR和inverse-PCR的方法克隆该突变体中T-DNA插入位置的旁邻序列,从而准确地将T-DNA定位到2号染色体上。基因表达分析显示,T-DNA插入位置附近的AK100376基因在J4突变体以及表型类似突变体neck leaf 1中的表达均被明显下调,可初步将其确定为与剑叶突变体表型相关的候选基因。     

扩展青霉碱性脂肪酶基因5'端侧翼区域的克隆与鉴定

应用衔接头PCR技术,扩增得到约2000 bp的扩展青霉碱性脂肪酶基因5’端侧翼区域的单一产物。对该产物测序并提交GenBank数据库(GenBank accession DQ677520),经序列比对分析,发现该序列具有真核启动子序列的基本结构特征,含有TATA盒、CAAT盒、GC盒等元件。将扩增得到的脂肪酶基因5’端侧翼序列,连接到含有绿色荧光蛋白(GFP)报告基因的质粒中,构建了一个重组表达质粒,转化大肠杆菌细胞。荧光显微观察大肠杆菌阳性转化子发出荧光,侧翼序列含有启动子功能得到确认。     

Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv. to rice,Oryza sativa L.

Oryza latifolia, a tetraploid wild relative of cultivated rice is an important source of resistance to bacterial blight (BB), the brown planthopper (BPH) and the whitebacked planthopper (WBPH). Interspecific hybrids were obtained between an elite breeding line (IR31917-45-3-2) of Oryza sativa (2n=24 AA) and O. latifolia Acc. No. 100914 (2n=48 CCDD). The crossability in F1 was 7.58% and it ranged from 0.11 to 0.62 in backcross generations. The F1 hybrid showed 2-6 II, 0-2 III, 0-1 IV and 22-32 I; the mean being 3.92 II + 0.11 III + 0.02 IV + 27.30 I per cell at diakinesis. Monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. latifolia were characterized based on morphology and isozyme banding pattern. The MAALs were designated as MAAL-1, MAAL-2, MAAL-4, MAAL-5, MAAL-6, MAAL-7, MAAL-8, MAAL-9, MAAL-10, MAAL-11 and MAAL-12. The female transmission rates of the alien chromosome varied from 4.4 to 35.5%, whereas 8 of the 11 MAALs transmitted the alien chromosome through the male gamete, the range being 1.7% (MAAL 10) to 11.9% (MAAL 12). Disomic progenies in BC3 and BC4 generations had complete resemblance to the O. sativa parent. Of the 2,295 disomic BC3F3 progenies, 309 showed introgression for resistance to BPH and 188 each for WBPH and BB resistance. Four plant progenies which were resistant to both BPH and WBPH were also resistant to BB race 2 of the Philippines. Nine of the 34 BC3F1 plants showed introgression for ten allozymes of O. latifolia, such as Est5, Amp1, Pgi1, Mdh3, Pgi2, Amp3, Pgd2, Est9, Amp2 and Sdh1, located on 8 of the 12 chromosomes. Alien introgression was also detected for morphological traits such as long awns, earliness, black hull, purple stigma and apiculus. Abnormal plants with many wild-species traits suddenly appeared in normal disomic progenies. These plants showing instability and abnormal segregation behaviour are being investigated for the activation of transposons.     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Regulation and function of mammalian DNA methylation patterns: a genomic perspective

Mammalian DNA methyltransferases (DNMTs) establish and maintain genomic DNA methylation patterns that are required for proper epigenetic regulation of gene expression and maintenance of genome stability during normal development. Aberrant DNA methylation patterns are implicated in a variety of pathological conditions including cancer and neurological disorders. Rapid advances in genomic technologies have allowed the generation of high resolution whole-genome views of DNA methylation and DNA methyltransferase occupancy in pluripotent stem cells and differentiated somatic cells. Furthermore, recent identification of oxidation derivatives of cytosine methylation in mammalian DNA raises the possibility that DNA methylation patterns are more dynamic than previously anticipated. Here, we review the recent progress in our understanding of the genomic function and regulatory mechanisms of mammalian DNA methylation.     

Walking into the unknown: a 'step down' PCR-based technique leading to the direct sequence analysis of flanking genomic DNA

We describe a novel and efficient PCR-based technique for walking into unknown flanking genomic DNA without recourse to protracted laborious library screening for overlapping sequences. This two component 'hot start' and 'step down' PCR method uses 6x1 microg of genomic DNA (ca 20kb in length) restricted with six different endonucleases and ligated to adaptors with the inclusion of two further restriction enzymes to prevent self-ligation. This allowed us to walk, in a single step, up to 6kb into flanking DNA and gave sufficient PCR products for up to 200 different walking experiments. This technology enabled us to clone and characterize the previously elusive 5' sequence of the barley powdery mildew chitin synthase gene, BgChs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene.     

High transformation frequency of tobacco and rice via Agrobacterium-mediated gene transfer by flanking a tobacco matrix attachment region

Nuclear matrix attachment regions (MARs) are suggested to regulate chromatin structure and influence the expression of flanking genes. Our previous study showed that TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro and increase transgene expression in vivo. Here, we investigated the role of TM2 MAR in improving transformation frequency of Agrobacterium -mediated transformation in tobacco and rice. The gusA reporter gene flanked by TM2 MAR in pBI121 and pCAMBIA-1301 vectors was controlled by a constitutive promoter or a photosynthetic tissue-specific promoter. The presence of TM2 MAR in different expression cassettes significantly increased the numbers of kanamycin-resistant tobacco shoots, hygromycin-resistant rice calli and shoots. Seeds from the independent transgenic lines with TM2 MAR can germinate normally on the medium containing 500 mg l⁻¹ kanamycin, whereas none of the seeds from the transgenic lines without TM2 MAR survived. Furthermore, RNA gel blot analysis revealed that nptII messenger RNA levels are more abundant in the independent transgenic lines with TM2 MAR than in the lines without TM2 MAR. Altogether, these data reveal a possible mechanism that TM2 MAR improves the transformation frequency by increasing nptII gene expression.     

Genome‐wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice

We conducted genome‐wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3‐binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3‐associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.     

转基因水稻中外源基因的荧光原位杂交（FISH）分析

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a transferred barnase-psl DNA sequence onto chromosomes in 8 rice transgenic plants. All the tested rice transgenic lines showed hybridization signals on the middle and terminal regions of chromosome arms except for those close to centromeres. In two lines, two different integration sites were identified, and the other lines showed only one integration site. With the aid of Southern analysis and expression detection, we found that the barnase tended to show a higher level expression in the lines whose integration sites near the distal regions of chromosomes, while the expression level became lower in the lines whose integration sites near the centromeres. This result suggested a possible relationship between chromosomal location of transgenes and the expression level. However it showed no obvious relationship between copy numbers and expression levels. In most cases, the results of multi-color FISH showed that barnase-ps1 always integrated at the same position on the chrmosome as the reporter genes(pHctinG).