Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

Chromosome microdissection by laser microbeam, chromosomal fragment isolation and amplificationin vitro in barley (Hordeum vulgare L.)

A method for microdissection, isolation and amplification of plant chromosomal fragments using laser microbeam and a glass microneedle was established. Firstly, 7H chromosome of barley (Hordeum vulgare L.) was dissected by Nd: YAG laserbeam with suitable parameters and the fragment comprising a satellite was isolated with a glass microneedle which was fixed on a micromanipulator. Then, the chromosomal fragment DNA was amplified by LA_PCR (linker adaptor PCR) for two rounds. The size of the DNA fragments of PCR products varied from 500-3 000 bp and the PCR products originated from the genome of barley were verified by Southern hybridization. Compared with previous reports, there are some advantages in this research. The performance is easier, the dissection is more precise and the cost is low. It also permits efficient amplification with only one single chromosome fragment. Laser microbeam_glass microneedle method may be useful in the microdissection of special chromosome regions, especially in plants with middle or small chromosomes.     

Cloning and characterization of a C genome-specific repetitive sequence inAegilops caudata L.

pAeca212 is 204 bp in length, and the G + C content is 51%. It disperses on all seven chromosome pairs ofAegilops caudata except centromeres and secondary constrictions. Compared with the 316893 DNA sequences registered in Genbank/EMBL/DDJB/PDB, pAeca212 is a new C-genome specific repetitive sequence. The results of genomic specificity analysis of pAece212 show that there are no hybridization signals detected in all donor Poaceae plants except in rye. pAeca212 is a very useful molecular marker in the study of the origin of Triticeace and the detection of C chromatin in wheat background.     

Physical location of riceGm-6, Pi-5(t) genes inO. officinalis with BAC-FISH

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. offi-cinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72 + 2.62 for 24E21 and 54+ 5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some ho-molgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.     

Construction of the primary physical map of rice chromosome 12

A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ∼16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome. Foundation item: Supported by Rockefeller Foundation Biography: FU Bin-Ying (1965-), male, Ph. D. candidate, Reseach direction: plant molecular genetics.     

Speciation- induced heritable cytosine methylation changes in polyploid wheat

To study possible epigenetic changes accompanying polyploid speciation, genomic DNA from natural polyploid wheats and their putative diploid progenitors were digested with a pair of isoschizomers Hpa II / Msp I and hybridized to 21 different types of low-copy DNA sequences. It was found that cytosine methylation changes were abundant in natural polyploid wheats after their speciation. The hybridization of the same set of sequences to a synthetic hexaploid wheat along with its parental lines indicated that the extensive DNA methylation changes already existed in the early generations (S5, S6 and Sy) of this plant. Moreover, the high similarity of the changed restriction fragment length polymorphism (RFLP) patterns among three randomly chosen individual plants suggested that the methylation changes occurred even earlier, and/or were of a nonrandom nature. The changed patterns were stably inherited in the three successive selfed generations. Though methylation changes are probably a genome-wide occurrence, they appeared to be confined to the specific types of DNA sequences. The possible implications of the rapid and extensive cytosine methylation changes for several attributes of allopolyploid genome evolution, such as genetic diploidization and gene diversification, are discussed .     

石蒜单染色体的显微分离及体外扩增

建立了石蒜单染色体的分离及体外扩增的方法。石蒜根尖经卡诺固定液固定后，再用果胶酶和纤维素酶酶解处理，制得标本，在倒置显微镜下，用自制的毛细管针挑取目的的染色体。将分离的石蒜染色体放入0．2mLEppedorf管中，经蛋白酶K处理后，进行DOP－PCR扩增，获得DNA片段。琼脂糖凝胶电泳显示扩增产物的长度大约为100－5000bp。此法为构建石蒜单染色体基因组库和筛选其特异性探针奠定了基础。     

菠菜性别差异NUPTs序列的克隆及分析

质体DNA向核基因组转移能够形成核质体DNA,核质体DNA序列的插入是推动动植物基因组及染色体组演化的重要动力.但是核质体DNA的插入和植物性染色体起源及演化之间的关系仍不清楚.以雌雄异株植物菠菜为材料,利用基因组消减杂交技术筛选分离菠菜雌雄基因组差异的NUPTs序列,并进行验证和分析.结果表明,从构建的菠菜消减杂交文库中共得到39条长度为75~308 bp的雌雄差异序列,其平均长度约为154 bp.对获得的序列进行Blastn同源比对发现了12条序列为叶绿体基因组来源序列,这些序列与菠菜叶绿体基因组相似度在98%以上,说明所获得的差异片段为核质体DNA序列.将筛选出的核质体DNA序列进行进一步验证后获得了两个稳定的长度分别为146 bp和199 bp的雄性偏好核质体DNA序列,说明所获得的两个NUPTs序列在菠菜雄性基因组中有更多的累积.     

LA-PCR法制备赤麂Y2涂染探针与原位杂交

应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR（linker-adaptor PCR）扩增后用DIG标记制备探针,然后用Southern blot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明：用该法制备的涂染探针是成功的,原位杂交也获得了阳性结果,可以初步验证毛冠鹿中的Y染色体.     

蜘蛛拖牵丝蛋白基因转家蚕表达质粒的构建

利用DNA重组技术对络新妇蛛(Nephila clavipes)拖牵丝蛋白基因MaSp1高度重复序列进行多次重组,人工构建成1.6 kb的蜘蛛拖牵丝蛋白人工基因Sil-E,DNA序列分析证明了人工基因序列的正确性.将家蚕L链基因启动子片段、L链cDNA、L链基因终止子融合在一起,构建成丝腺特异性表达单元.再与Sil-E融合构建成蜘蛛拖牵丝蛋白基因家蚕丝腺特异表达单元.将该表达单元克隆到转座子piggyBac的转基因载体中,获得了蜘蛛拖牵丝蛋白转基因表达载体.采用显微注射法将其与辅助质粒共导入到家蚕蚕卵中.筛选转基因阳性个体,经PCR和Southern杂交鉴定,结果表明目的基因整合到家蚕基因组中,为进一步研究家蚕作为生物反应器表达蜘蛛拖牵丝蛋白基因奠定了基础.     

Construction and use of human chromosome jumping libraries from NotI-digested DNA

A basic difficulty in the molecular analysis of genes identified by mutations in the mammalian genome is the need to cover genetic distances corresponding to several hundred kilobases or more by molecular techniques like chromosome walking. In chromosome jumping, this limitation is overcome by the deletion of all but the extreme ends of large DNA molecules before cloning. We describe here the construction and characterization of a NotI 'jumping library' from human DNA. To characterize this library, random clones were analysed by restriction mapping. Clones carrying unique end fragments were characterized further by hybridization to Southern blots of NotI-cleaved human DNA separated on pulsed field gradient (PFG) gels. As a first step in a directional walk, the library was screened with a clone containing a NotI site cleaved in genomic DNA ('NotI linking clone') localized to the distal third of the short arm of human chromosome 4 (A.-M.F. & T.P., unpublished data). Starting and end points of two identified clones were positioned within a restriction map covering 850 kilobases.     

Studies inin situ PCR detected by the BrdU antibody technique

Using the BrdU antibody technique followed by an immuno-chemical staining (BAT), the amplification of DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products orin situ hybridization with PCR products on slides, the amplified target DNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differences in the sensitivity and efficiency between BAT and dig-11-dUTP labeling in cellin situ PCR were discussed. Zhang Xiyuan: born in Oct. 1935, Professor, Current research interest in Cellular and Molecular Biology Supported by the National Natural Science Foundation of China     

PCR amplification of Sox genes inMisgurnus anguillicaudatus andParamisgurnus dabryanus (Cypriniforms; Cobitidae)

Using the primers which specially amplify the conservative motif of Human SRY gene, we studied the PCR amplification of Sox genes in genomic DNA of two species of mud loach:Misgurnus anguillicaudatus andParamisgurnus dabryanus. Four bands with the length of 200,550,940 and 1000 bp respectively, were presented in the PCR products ofMisgurnus anguillicaudatus. Three bands with the length of 200,550 and 900 bp were presented in that ofParamisgurnus dabryanus. Southern blotting results indicated that the 200 and 550 bp bands are specially positive. There is no difference between male and female individuals as well as between these two species. Chang zhongjie: Born in Nov. 1965, Ph. D. graduate student     

Genomic and genic sequence variation in synthetic hexaploid wheat (AABBDD) as compared to their parental species

In order to understand the genomic changes during evolution of hexaploid wheat, two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat (AABB) and paternal diploid goat grass (DD) were used for DNA-AFLP and single strand conformation polymorphism (SSCP) analysis to determine the genomic and genic variation in the synthetic hexaploid wheat. Results indicated that more DNA sequences from paternal diploid species were eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat, suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially. However, sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP, which indicated that much less variation in the genic regions occurred in the synthetic hexaploid wheat, and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences. Our results also indicated that sequence variation in 4 genes can be detected in hybrid F1, which suggested that this type of sequence variation could be resulted from distant hybridization. It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long arm of chromosome 2D, which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.     

Genomic and genie sequence variation in synthetic hexaploid wheat(AABBDD)as compared to their parental species

In order to understand the genomic changes during the evolution of hexaploid wheat,two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat (AABB) and paternal diploid goat grass(DD)were used for DNA-AFLP and single strand conformation polymorphism (SSCP) analysis to determine the genomic and genie variation in the synthetic hexaploid wheat.Results indicated that more DNA sequences from paternal diploid species wen eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat,suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially.However,sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP.which indicated that much less variation in the genie regions occurred in the synthetic hexaploid wheat.and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences.Our results also indicated that sequence variation in 4 genes can be detected in hybrid F1.which suggested that this type of sequence variation could be resulted from distant hybridization.It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long alTll of chromosome 2D,which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.     

Genomic and genic sequence variation in synthetic hexaploid wheat （AABBDD） as compared to their parental species

In order to understand the genomic changes during the evolution of hexaploid wheat, two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat （AABB） and paternal diploid goat grass （DD） were used for DNA-AFLP and single strand conformation polymorphism （SSCP） analysis to determine the genomic and genic variation in the synthetic hexaploid wheat. Results indicated that more DNA sequences from paternal diploid species were eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat, suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially. However, sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP, which indicated that much less variation in the genic regions occurred in the synthetic hexaploid wheat, and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences. Our results also indicated that sequence variation in 4 genes can be detected in hybrid F1, which suggested that this type of sequence variation could be resulted from distant hybridization. It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long arm of chromosome 2D, which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.     

小麦液泡质子焦磷酸酶基因EST的克隆与分析

通过RT-PCR的方法在矮苏3小麦的总cDNA中克隆到一个与大麦液泡质子焦磷酸酶基因(VP)高度同源的EST。以该序列为基础,利用生物信息学的方法构建了一个编码小麦VP蛋白的全长EST重叠群,长2764bp,其包含一个长2355bp的完整开放读码框(ORF),编码785个氨基酸多肽。通过Southern杂交,将该VP基因定位在小麦染色体的第7同源群上。     

野生一粒小麦着丝粒RCS1相关序列的克隆鉴定

用水稻着丝粒重复序列RCS1为探针 ,与 30 72个克隆进行菌落杂交 ,得到了 32个阳性克隆 ,用RCS1与拟斯卑尔脱山羊草着丝粒重复序列Tcs2 5 0为探针进一步筛选 ,在 32个RCS1相关的阳性克隆中任选 10个克隆进行点杂交 ,分别有 6个和 5个阳性克隆 .为了克隆RCS1相关片段 ,依据RCS1的序列设计了三对引物 ,将引物 3从上述阳性克隆中扩增的一个 5 4 3bp的片段克隆测序 ,发现与水稻RCS1部分片段达到约 83%的同源 ,与大麦的反转座子 (Ty3 gypsy)部分序列同源性达到了 92 % ,与节节麦中着丝粒的整合酶基因部分序列同源性达到了 96 % ,命名为TBRCS1.TBRCS1可能是野生一粒小麦着丝粒区的组成部分