Construction and initial analysis of five Fosmid libraries of mitochondrial genomes of cotton （Gossypium）

Cotton （Gossypium） is an important crop providing textile fiber and edible oil. To gain the insights into mechanism of the cyto- plasmic male sterility （CMS） inheritance, we constructed five fosmid libraries of mitochondrial genomes from mitotype of G. harknessii Brandegee. （one CMS line and its restorer）, mitotype of G. hirsutum L. （one CMS line and its maintainer）, and G. bar- badense L. The numbers of the clones in these libraries ranged from 1152 to 2016 with an average insert size of 36.2 to 38.4 kb, equivalent to 70-119.3 mitogenomes. The libraries were screened with 28 markers derived from the conservative sequences and yielded 22, 19, 26, 21, and 23 positive clones, respectively. These positive clones were used to construct the physical map of G. harknessii Brandegee. CMS line and G. barbadense L. mitogenomes that shared six syntenis regions. A total of 30 genes in nine clusters showed conservative and had high similarity with those in the mitochondrial genomes of cotton, Carica papaya, Cucur- bita pepo and Nicotiana tabacum. Further investigation indicated that gene rrn26 had two copies in all five cotton mitogenomes, while genes atpl, rrn5 and rrn18 had two copies only in G. barbadense L. The positive clones and physical map are considered being useful resources in cotton genomics research.     

中国美利奴细毛羊BAC文库的质量评价

从文库的平均插入片段大小、文库的覆盖率、文库克隆的稳定性对中国美利奴细毛羊BAC文库进行了质量评价.统计结果表明,文库的平均插入片段大小约为133kb, 非重组克隆占3.2%,文库在理论上约有8倍绵羊基因组覆盖率,从该文库中找到任意单拷贝序列的概率为99.93%.连续多次传代培养实验证实文库克隆的遗传稳定性好.     

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, 32 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.     

A five-fold pig bacterial artificial chromosome library: a resource for positional cloning and physical mapping

A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind III or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480 pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10 single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.     

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, 32 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.     

Screening and Insert Size Analysis of Candidate C-Genome BAC Clones from Brassica napus

Thirty-two C-genome specific candidate bacterial artificial chromosome (BAC) clones were successfully screened from the BAC library by four-dimensional PCR method with the primer pairs of 75 simple sequence repeat (SSR) markers located in the nine C-genome linkage groups of Brassica napus. The screened 32 BAC clones have an average insert size of 114.2 kb with a range of 30-190 kb. They are the first set of C-genome BAC clones screened from B. napus genomic BAC library. The average insert size of this set of BAC clones presented that the constructed BAC library had a high quality. This set of BAC clones can be used as markers to identify individual chromosomes of B. napus C-genome.     

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.     

Inheritance of the delayed gland morphogenesis trait in Australian wild species ofGossypium

Five Australian wild cotton species with the delayed gland morphogenesis trait, as well as G. arboreum, G. davidsonii and four different gland genotypes of G. hirsutum, Gl₂Gl₂Gl₃Gl₃, Gl₂Gl₂gl₃gl₃, gl₂gl₂Gl₃Gl₃, and gl₂gl₂gl₃gl₃, were used in this experiment and 10 interspecific hybrids were obtained by the crossing among them. According to the gland expression on the seeds and plants of the interspecific hybrids, the inheritance of the delayed gland morphogenesis trait of Australian wild cotton species was opened out as follows: (ⅰ) the inheritance of the delayed gland morphogenesis trait was almost the same among the 5 Australian wild cotton species, and the gene or genes which controlled this trait may be located in the same loci. (ⅱ) The glandless seed trait of the Australian wild cotton species was dominant over the glanded seed trait of G. arboreum, a genome A species, and the seeds of interspecific hybrid F₁ between them were glandless. However, it was recessive over the glanded character of genome D species, G.davidsonii, and their F₁ was a typical glanded one. (ⅲ) The glandless seed trait of the Australian wild cotton species was recessive or incomplete dominant over the glanded cotton but dominant over the glandless cotton of G. hirsutum, and the glandless genes (gl₂gl₂gl₃gl₃) of upland cotton had great weakening effect on the glanded plant trait of the Australian wild cotton species on the other hand. For the two main glanded genes of upland cotton, the delayed gland morphogenesis trait of the Australian wild cotton species was dominant epistatic over glandless genes, gl₂gl₂gl₃gl₃, and one of the glanded genes, Gl₂Gl₂, but was recessive epistatic over the other glanded gene, Gl₃Gl₃. Therefore, it is much convenient to use Gl₂Gl₂gl₃gl₃ as the upland cotton parent in the interspecific hybridization and backcrossing afterward, in order to produce the upland cotton germplasm with glandless seeds and glanded plant trait.     

厚壳贻贝足腺cDNA文库的构建及部分EST序列分析

为获得厚壳贻贝足腺组织的EST序列标签以及可能的功能基因序列,以pCMVsport6质粒为载体构建了高质量的厚壳贻贝足腺组织cDNA文库,文库滴度为1.31×107pfu/mL,重组率为98%,平均插入片段为1.3kb.通过对文库部分克隆的序列测定,获得了11个新基因和17个功能基因,其中包括部分与厚壳贻贝足丝相关的基因.厚壳贻足腺组织cD-NA文库的成功构建为将来筛选与厚壳贻贝足丝蛋白相关的新基因,系统研究其黏附机制奠定了基础.     

Construction and characterization of a bacterial artificial chromosome library of peach

This report briefly describes the construction and characterization of a peach [ prunus persica (L.) Batch] Var. Jingyu bacterial artificial chromosome (BAC) library. The variety Jingyu has many important agronomic characters of stone fruits, and it is a main parent in Chinese peach breeding. After cloning of the high molecular weight peach DNA into pBeloBAC 11, we obtained over 22 000 recombinant clones. The BAC library has an average insert size of 95 kb and represents approximately 7 times peach haploid genome equivalents. After being screened with two randomly amplified polymorphic DNA markers, W4 and P20, which are linked to yellow flesh and nectarine genes of peach respectively, ten positive clones have been detected. This library is very useful for map-based cloning of peach genes and physical mapping of peach genome.     

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50—400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.     

活性污泥宏基因组Fosmid文库的构建

大插入片段宏基因组文库的构建是开发大片段目的基因及分析其结构与功能的基础.文中分别采用十六烷基三甲基溴化铵(CTAB)法、试剂盒及琼脂糖包埋法提取活性污泥宏基因组DNA,其中琼脂糖包埋法获得的DNA片段大于23kbp,利用此DNA成功构建了以pCC1FOS为载体的Fosmid文库,该文库含有5280个克隆,平均插入片段长度为35～40 kbp,共包含约200 Mbp的宏基因组DNA.从此文库中随机挑选200个克隆,利用活性筛选方法快速筛选到了1个含有淀粉酶的阳性克隆,表明活性污泥Fosmid文库可用于功能基因的活性筛选,具有开发新基因的潜力.     

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.     

Genomic cloning and sequencing of betaine aldehyde dehydrogenase gene in spinach

A genomic library derived from leaves of spinach was constructed with the λGem11_BamHI Arms as the vector. The library was screened using the BADH cDNA of mountain spinach as a probe and six positive clones were obtained through three rounds of screening. One of the positive clones named D, which was hybridized with the 5′600 bp fragment of mountain spinach BADH cDNA, was selected and further analyzed. The size of the insert in clone D was about 12 kb. 8 856 nucleotides of the insert were sequenced which contained 2 459 nucleotides of 5′ noncoding region, 6 111 nucleotides of the complete sequence of the BADH gene, and 286 nucleotides of a 3′ noncoding region. The result of sequence analysis indicated that the BADH gene contained 14 introns and the junction sequences at splicing sites followed the GT_AG rule basically.     

Construction and characterization of a normalized whole-life-cycle cDNA library of rice

A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies.For studying the functions of rice genes on a large scale,a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63,an elite restorer line for a number of rice hybrids that are widely cultivated in China,This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages.For normalization,the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library.This library consists of 62000 clones with an average insert length about 1.4kb.Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes.Sequencing of 10750 cDNA clones of this library reveals 6399 unique EST s(expressed sequence tags),indicating that the non-redundancy of the library is about 59.5%.This library has been used to make cDNA microarrays for functional genomic studies.     

Evaluation of the introgressed lines and screening for elite germplasm in Gossypium

THERE ARE 51 SPECIES IN THE GENUS OF GOSSYPIUM[1]. EXCEPT UPLAND COTTON CULTIVARS, WILD CULTIVARS, SPECIES AND RACE HAVE ABUNDANT GENETIC POLYMORPHISMS DUE TO THEIR VARIOUS ENVIRONMENTAL DISTRIBUTION AND LONG-TERM NATURAL SELECTION. THEREFORE, THEY POSSESS LOTS OF EXCEL- LENT GENES THAT CAN BE USED TO EXPLOIT POTENTIAL TRAITS, SUCH AS DROUGHT RESISTANCE, DISEASE AND INSECT …     

盐藻(Dunaliella viridis)细菌人工染色体文库的构建和鉴定

盐藻具有极强的耐盐能力,是研究植物耐盐机制的模式系统.为了对其耐盐机制进行深入的 研究,以pCC1BAC为载体,构建了盐藻的细菌人工染色体（BAC）文库.该文库共有9 216 个转化子,插入片段平均长度为55 kb,以单克隆形式保藏在96块96孔板中,并建立了四维P CR基因筛选体系,可以通过4轮PCR快速筛选获得阳性单克隆.根据本实验室分离到的两个盐 藻基因的cDNA序列（ DvSPT2和DvTPSP ）设计引物,通过PCR从该文库中各筛到4个阳性BA C克 隆,说明该文库能有效用于分离盐藻基因的基因组序列,据此推测该文库约覆盖4倍盐藻基 因组序列.     

Construction of a bacterial artificial chromosome library of S-type CMS maize mitochondria

In order to isolate mitochondrial genes easily, we have developed a new method to construct S-type CMS maize mitochondrial gene library by means of embedding mitochondria and enzymatic digesting mitochondriain situ, preparing mtDNA by electrophoresis, digesting LMP agarose with β-agarase, using BAC vector and electroporation. About 2 500 white clones of Mo17 CMS-J mitochondrial gene library were obtained with the average size of 18.24 kb, ranging from 5 to 40 kb, 63.6% inserts came from mitochondrial genome and represented 48 × mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. It is helpful to elucidating the maize mitochondrial genome structure and mechanism of S-type CMS, and may give some valuable reference to the construction of other plant mitochondrial genome library.     

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomic in situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted from Gossypium species as probe. With twice FISH to the same mitotic cell of G herbaceum or G hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs from G hirsutum and Graimondii mitotic images were all terminal types. Four and two GISH-NORs from G herbaceum (var. africanum) were terminal and centromere types, respectively. Six GISH-NORs in G hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image of G raimondii with its own gDNA as probe. From mitotic image of G herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-wholerecovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes in Gherbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.