Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

Physical location of the ricePi-5(t), Glh andRTSV genes by ISH of BAC clones

Mapping of low or single-copy sequences on plant chromosomes has proven difficult because of very low frequency of signal detection. Rice BAC library is being used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two biotin-labeled rice BAC clones closely linked to a rice blast resistance, green leafhopper resistance and tungro spherical virus resistance gene,Pi-5(t), Glh, RTSV, werein situ hybridized to rice chromosomes. They were located on the long arm and short arm of chromosome 4 with FL value of 40% and 100% respectively. The frequency of signal detection reached 46.8% and 59.2%. The signal location were consistent with the selective marker on rice saturated molecular map. The results demonstrated the advantages to locate BAC clones to chromosomes byin situ hybridization and will facilitate the rice low or single-copy gene location by using the BAC library. Supported by the National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Department of the People's Republic of China Yan Huimin: born in 1964, Lecturer     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

Ipi-t,Ipi-3(t)在水稻中的物理定位及水稻第12染色体的识别

选用与水稻12染色体上Ipi-t,Ipi-3(t)和Pi-4(t)及着丝粒连锁的RFLP标记RZ670对水稻进行了荧光原位杂交。清楚显示了第12染色体着丝粒所在的位置,为水稻染色体的准确识别提供了一种新的方法。     

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.     

Isolation and identification of the three rice monotelosomics

In order to obtain rice monotelosomic, the progeny of 24 telotrisomics, derived from an indica rice variety, Zhongxian 3037, were screened. The variants that differed morphologically from the diploids and the original primary trisomics as well as the telotrisomics were collected for cytological identification. The variants with 24 chromosomes were selected according to the prometaphase chromosomes. From these variants, three monotelosomies with one chromosome arm deletion in each were verified by fluorescence in situ hybridization （FISH） using a rice centromeric BAC clone of 17p22 as a marker probe. The three monotelosomics were derived from telotrisomic 1S, 4L and 11L, respectively. Further identification was conducted on the prometaphase or pachytene chromosomes of the three variants, which were probed with the same centromeric BAC clone together with the corresponding chromosome arm specific makers, a0059H02 （on the short arm of chromosome 1）, a0034E24 （on the long arm of chromosome 4）, and a0071H11 （on the long arm of chromosome 11）. The results indicated that the telocentric chromosomes in the three monotelosom. ics were derived from their respective corresponding telotrisomics. According to the telocentric chromosomes of the variants, they were monotelosomic 1S （one long arm of chromosome 1 was lost）, monotelosomic 4L （one short arm of chromosome 4 was lost） and monotelosomic 11L （one short arm of chromosome 11 was lost）, respectively.     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.     

FISH analysis of the integration patterns in transgenic rice co-transformed by microprojectile bombardment

Using multi-color fluorescencein situ hybridization (FISH), we localized transferredbarnase-ps1 andpHctinG DNA sequences onto chromosomes of two transgenic rice plants, named Q12 and Q13, both of which were produced by micro-projectile bombardment. In both Q12 and Q13, each detected cell showed 2–3 signal spots on their chromosomes respectively. The signals of bothbarnase-ps1 andpHctinG were mostly detected in the adjacent chromosomal sites in which their signals were overlapped and could be recognized by the signal color on the metaphase chromosomes. Fiber FISH further demonstrated that the multiple copies in each of the two DNA sequences distributed adjacently on the DNA fiber in Q13. Combined with the results of Southern hybridization, the possible integration patterns in transgenic rice co-transformed by micro-projectile bombardment have been discussed.     

着丝粒串联重复序列RCS2对亚洲栽培稻和非洲栽培稻的比较分析

为了揭示中高度重复序列在同为AA基因组的亚洲栽培稻和非洲栽培稻基因组中的差异以及重复序列在.栽培稻种的分化过程中可能起到的作用,利用水稻着丝粒串联重复序列RCS2作为探针分别对籼稻广陆矮4号、粳稻日本晴和非洲栽培稻的体细胞染色体进行荧光原位杂交(FISH)实验,并对其核型进行同源性聚类和比较分析,杂交结果显示:RCS2序列位于在3种栽培稻染色体组中,RCS2序列位于每条染色体的着丝粒位置,但有不同的分布特点,表明该3种栽培稻基因组的RCS2序列有不同的进化方向.探讨了RCS2序列结合Cot-1 DNA FISH方法对水稻染色体组进行核型分析的可行性和优势.     

利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.     

抗稻瘟病水稻BAC文库的构建与鉴定

水稻是一种重要的粮食作物，同时也是一种重要的单子叶植物模式.稻瘟病是水稻生长中的一种严重病害，因此分离和克隆新的稻瘟病抗病基因具有重要的应用价值.以一个抗稻瘟病农家种水稻为材料，以plndigoBAC5为载体，构建了细菌人工染色体（BAC）文库.该文库共有90 000个转化子，插入频率99％，插入片段平均长度为105 kb，由此推测这个文库覆盖水稻基因组约20倍.利用其中的45 000个转化子建立了4维PCR筛选体系，并且通过4维PCR筛选体系，筛选获得了7个含水稻分蘖基因（MOC1）的阳性克隆.因此该文库是一个高质量、高覆盖率的水稻BAC文库，能有效用于目的基因的分离.     

Construction of a BAC library from cucumber （Cucumis sativus L.） and identification of linkage group specific clones

A bacterial artificial chromosome （BAC） library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber （Cucumis sativus L.） inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions （SCAR） and seven simple sequence repeats （SSR） markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction （PCR）. Fifteen markers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

一个药用野生稻异源单体附加系在减数分裂时期的GISH分析鉴定

异源单体附加系是从亲缘关系较远或属间的一个物种单条染色体附加到另一个物种中．栽培稻珍籼97B与药用野生稻Hy18杂交与连续回交，在BC2后代中得到一个药用野生稻单体附加系．生物素标屺的药用野生稻总DNA作为探针，未标记的栽培稻总DNA封阻，对其异源单体系减数分裂染色体进行基因组原位杂交．FISH结果表明，在栽培稻（AA，2n=24）基因组中附加了一条药用野生稻染色体，并鉴定为第8号染色体．研究表明，药用野生稻异源单体附加系的建立为药用野生稻的基因组学和遗传学的研究提供一个新的操作平台．而GISH技术在水稻远缘杂交育种中是最准确有效的染色体鉴定方法．在水稻育种改良中具有重要应用前景．     

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     

利用DNA微卫星标记定位水稻的抗稻瘟病基因

利用回交育种中产生的回交群体结合前人的研究结果构建了Pil基因区域的局部分子标记连锁图，通过BC1F2家系的接种结果判断其基因型，将Pil定位在RFLP标记RZ536与SSR标记RM144之间，图距分别为9．7、6．8cm，从而建立了一套完整的以PCR为基础的分子标记辅助选择体系。     

克隆含有MT-Ⅰ和MT-Ⅱ基因的染色体DNA片段及其内切酶图谱的鉴定

用小鼠MT-ⅠcDNA作为探针,从129小鼠的基因组库中获得含MT-Ⅰ基因的DNA片段。从6.8×10⁵的噬菌斑中挑出4个阳性噬菌体克隆,分别命名为1-1, 2-1, 1-6和4-3。在这4个克隆中1-1和2-1的阳性信号更强些。应用插入片段的末端引物作为探针进行杂交结合部分酶切的方法,对这2个克隆的插入片段进行了限制性内切酶酶切图谱分析,确定了克隆1-1和2-1的酶切图谱及MT-Ⅰ基因在2个克隆DNA中的位置。并进一步发现和证明了在2个克隆中含有MT-Ⅱ基因。     

水稻第6染色体S5区重叠群构建、基因注释与OS-APH的克隆分析

以通过图位克降所获得的Cosmid克降R2119序列信息为基础，构建了一个长为586kb的粳稻第6染色体S5座位的BAC重叠群（JS5-BC）。通过生物信息学方法，对JS5-BC进行了基因注释，确定该区域含有46个基因位点。经功能预测，JS5-BC重叠群中仔在3个主要的基因家族，分别是木葡聚糖岩藻糖基转移酶、脂酶和黄素氧化还原酶。JS5-BC中注释基因的一个显著特点是功能相关基因密集存在，如与植物细胞壁的合成代谢相父的3个基因、与呼吸链能量代谢的黄素氧化还原酶的4个基因等都是如此。为了验证基因注释的可靠性，克降了广亲和品种Cp17品种的OSAPH基因的cDNA，并检测了该基因的表达模式，为注释基因提供了分子证据。     

Physical location ofHelminthosporium carbonum susceptibility gene hm1 by FISH of a RFLP marker umc119 in Maize

A fluorescencein situ hybridization (FISH) procedure was adopted to physically map a RFLP marker, umc119 near the centromere of the long arm of linkage group1 in maize. The hm1 gene (Helminthosporium carbonum susceptibility gene) was linked closely with the marker umc119. RFLP markers are very good landmarks for mapping genes. Therefore, we also determined the position of the gene hm1 on the chromosome based on the physical location of umc119. The disease induced by infection ofHelminthosporium carbonum is one of the serious maize diseases and it distributes in many countries including China. Hybridization sites were showed on 1 L (long arm of chromosome1) and 5 L. The percentage distance from centromere to the hybridization site was 22.86 on 1 L and 58.23 on 5 L the detection rate was about 12% for mitotic cells. In interphase nuclei five hybridized sites were detected. It demonstrated that umc119 was multiplicated sequences. FISH has more advantages overin situ hybridization (ISH) detected by DAB for increasing the detection ratio and contrast between chromosomes and hybridization signals. The ability to detect the hybridization signal of a small low copy DNA sequence is a very important key towards wide application of FISH for plant genome mapping. Supported by the National Natural Science Foundation and Doctorate Vesting Point Foundation of the Education Committee of China Li Lijia: born in 1967. Ph. D.     

不同栽培品种山茱萸药材中多糖的含量测定

采用硫酸-苯酚法测定不同栽培品种山茱萸药材中多糖的含量.7个栽培品种33份样品测定结果显示,我国山茱萸产区的5个主流品种:椭圆形果型、圆柱形果型、长圆柱形果型、长梨形果型、短圆柱形果型中多糖的含量差异较小,山茱萸多糖可以作为山茱萸药材的质量控制指标,为山茱萸药材的质量评价提供依据.     

转基因水稻与野生稻（Oryza nivara Sharma et Shastry）杂交后代种子萌发率

种子休眠性是野生植物重要的适应性状,通过种子萌发实验可以分析种子休眠性强弱.通过人工杂交获得转抗虫基因栽培稻与一年生普通野生稻三种组合的杂种,对杂种的F3代和F4代种子采用直接萌发、打破休眠后萌发、埋土15 d和30 d 4种不同处理来检测种子活力和萌发率.结果显示转基因杂种后代种子表现出较强的休眠性,转基因对种子活力和休眠性没有明显的影响,种子休眠性有随种子世代的增加而逐渐减弱的趋势,提示水稻转基因逃逸后有在野生稻群体中宿存和扩散的可能性,但这种可能性可能会随世代增加而下降.这为进一步研究水稻转基因逃逸风险提供了参考.