共查询到20条相似文献,搜索用时 15 毫秒
1.
Suzuki K Asakawa S Iida M Shimanuki S Fujishima N Hiraiwa H Murakami Y Shimizu N Yasue H 《Animal genetics》2000,31(1):8-12
A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences. 相似文献
2.
Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing 总被引:2,自引:0,他引:2
Yang TJ Yu Y Nah G Atkins M Lee S Frisch DA Wing RA 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2003,107(4):652-660
Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome. 相似文献
3.
Park MH Lee HJ Bok J Kim CH Hong ST Park C Kimm K Oh B Lee JY 《Journal of biochemistry and molecular biology》2006,39(4):418-425
A human bacterial artificial chromosome (BAC) library was constructed with high molecular weight DNA extracted from the blood of a male Korean. This Korean BAC library contains 100,224 clones of insert size ranging from 70 to 150 kb, with an average size of 86 kb, corresponding to a 2.9-fold redundancy of the genome. The average insert size was determined from 288 randomly selected BAC clones that were well distributed among all the chromosomes. We developed a pooling system and three-step PCR screen for the Korean BAC library to isolate desired BAC clones, and we confirmed its utility using primer pairs designed for one of the clones. The Korean BAC library and screening pools will allow PCR-based screening of the Korean genome for any gene of interest. We also determined the allele types of HLA-DRA and HLA-DRB3 of clone KB55453, located in the HLA class II region on chromosome 6p21.3. The HLA-DRA and DRB3 genes in this clone were identified as the DRA*010202 and DRB3*01010201 types, respectively. The haplotype found in this library will provide useful information in future human disease studies. 相似文献
4.
A bacterial artificial chromosome library for sugarcane 总被引:10,自引:0,他引:10
J. P. Tomkins Y. Yu H. Miller-Smith D. A. Frisch S. S. Woo R. A. Wing 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):419-424
Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits
in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate
cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The
library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average
insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were
gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm2 filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of
the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of
less than 1%. To demonstrate the library’s potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored
to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization
signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for
insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison
with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted,
and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones
were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene.
Received: 12 September 1998 / Accepted: 12 March 1999 相似文献
5.
S. Allouis X. Qi S. Lindup M. D. Gale K. M. Devos 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,102(8):1200-1205
A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with
an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening
by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset
of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified
between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was
estimated to be less than 1% by hybridisation with a rice chloroplast probe.
Received: 30 January 2000 / Accepted: 16 October 2000 相似文献
6.
Y. Yu J. P. Tomkins R. Waugh D. A. Frisch D. Kudrna A. Kleinhofs R. S. Brueggeman G. J. Muehlbauer R. P. Wise R. A. Wing 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,101(7):1093-1099
Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for
positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library
for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert
size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated
an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing
a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a
Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm2 filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening
the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe.
A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions
of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance
primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural
genomic applications in barley.
Received: 20 September 1999 / Accepted: 25 March 2000 相似文献
7.
Jian-Mei Yin Wang-Zhen Guo Tian-Zhen Zhang 《植物学报(英文版)》2006,48(2):219-222
A bacterial artificial chromosome (BAC) library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker. 相似文献
8.
For molecular and cytogenetic studies, two partial bacterial artificial chromosome (BAC) libraries of the garlic cultivar Allium sativum L. 'Danyang' were constructed using high molecular weight (HMW) garlic DNA, the pBAC1-SACB1 vector, and the pIndigoBAC536 vector. The average insert size of the BAC library was about 90 kb. The sequence compositions of the BAC clones were characterized by Southern hybridization with garlic genomic DNA and a repetitive sequence clone of garlic. Two BAC clones with weak signals (thus implying mostly unique sequences), GBC2-5e and GBC2-4d, were selected for FISH analysis. FISH analysis localized the GBC2-5e (approximately 100 kb) BAC clone on the long arm of garlic chromosome 7. The other BAC clone, GBC2-4d (approximately 110 kb), gave rise to discrete FISH signals on a mid-size early metaphase chromosome. The FISH screening with BAC clones proved to be a useful resource for molecular cytogenetic studies of garlic, and will be useful for further mapping and sequencing studies of important genes of this plant. 相似文献
9.
Construction of a hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome library for cloning genes for stripe rust resistance. 总被引:2,自引:0,他引:2
A hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome (BAC) library was constructed for cloning Yr5 and other genes conferring resistance to stripe rust (Puccinia striiformis f. sp. tritici). Intact nuclei from a Yr5 near-isogenic line were used to isolate high molecular weight DNA, which was partially cleaved with HindIII and cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consisted of 422,400 clones arrayed in 1100 micro-titer plates (each plate with 384 wells). Random sampling of 300 BAC clones indicated an average insert size of 140 kb, with a size range from 25 to 365 kb. Ninety percent of the clones in the library had an insert size greater than 100 kb and fewer than 5% of the clones did not contain inserts. Based on an estimated genome size of 15,966 Mb for hexaploid wheat, the BAC library was estimated to have a total coverage of 3.58x wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing an Yr5 locus-specific marker (Yr5STS7/8) were successfully selected by PCR screening of 3-dimensional BAC pools. The results demonstrated that the T. aestivum BAC library is a valuable genomic resource for positional cloning of Yr5. The library also should be useful in cloning other genes for stripe rust resistance and other traits of interest in hexaploid wheat. 相似文献
10.
Ling Liu Wei Li Yong Peng Runlin Z. Ma 《Biochemical and biophysical research communications》2010,391(2):1280-1284
In a search for genes affecting intramuscular fat deposition, we constructed a bacterial artificial chromosome (BAC) library for the whole genome of Rongchang pig, a domestic Chinese swine breed. The library consisted of approximately 192,000 clones, with an averaged insert size of 116 kb. Frequency of non-insert clone of the BAC library was no higher than 1.8%, based on estimation of 220 BAC clones randomly selected. We estimated the coverage of the library to be more than seven porcine genome equivalents. Subsequent screening of the BAC library with a three-step PCR procedure resulted in identification of seven candidate genes that were potentially involved in intramuscular fat deposition. The number of positive BAC clones ranged from 2 to 4 for each of the seven genes. One positive clone, containing the lipin1 gene, was fully sequenced by shotgun method to generate 118,041 bp porcine genomic sequences. The BAC clone contained complete DNA sequence of porcine lipin1 gene including all the exons and introns. Our results indicate that this BAC library is a useful tool for gene identification and help to serve as an important resource for future porcine genomic study. 相似文献
11.
A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2 总被引:2,自引:0,他引:2
D. M. O’Sullivan P. J. Ripoll M. Rodgers K. J. Edwards 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(2-3):425-432
We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in
European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving
approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection
for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation
efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of
clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened
by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow
rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST)
markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised
to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.
Received: 30 August 2000 / Accepted: 6 December 2000 相似文献
12.
Xiaojun Zhang Yang Zhang Chantel Scheuring Hong-Bin Zhang Pin Huan Bing Wang Chengzhang Liu Fuhua Li Bin Liu Jianhai Xiang 《Marine biotechnology (New York, N.Y.)》2010,12(2):141-149
The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species. 相似文献
13.
Buitkamp J Kollers S Durstewitz G Welzel K Schäfer K Kellermann A Lehrach H Fries R 《Animal genetics》2000,31(6):347-351
A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de). 相似文献
14.
Vilanova S Romero C Abernathy D Abbott AG Burgos L Llacer G Badenes ML 《Molecular genetics and genomics : MGG》2003,269(5):685-691
To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves ( Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.Communicated by R. Hagemann 相似文献
15.
Tomkins JP Miller-Smith H Sasinowski M Choi S Sasinowska H Verce MF Freedman DL Dean RA Wing RA 《Microbial & comparative genomics》1999,4(3):203-217
Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome. 相似文献
16.
Two-dimensional screening of the Wageningen chicken BAC library 总被引:10,自引:0,他引:10
Richard P.M.A. Crooijmans Julia Vrebalov Rosilde J.M. Dijkhof Jan J. van der Poel Martien A.M. Groenen 《Mammalian genome》2000,11(5):360-363
We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken
genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial
HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb
was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate,
and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken
BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was
obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker
present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the
number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool
for positional cloning and for comparative mapping studies.
Received: 26 October 1999 / Accepted: 6 January 2000 相似文献
17.
Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus 总被引:2,自引:0,他引:2
Jang S Zhou F Xia L Zhao W Cheng H Zhou R 《Biochemical and biophysical research communications》2006,348(2):775-780
A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes. 相似文献
18.
Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP 总被引:2,自引:0,他引:2
Babcock DA Wawrik B Paul JH McGuinness L Kerkhof LJ 《Journal of microbiological methods》2007,71(2):156-161
It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed. 相似文献
19.
Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. 总被引:30,自引:1,他引:29 下载免费PDF全文
The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants. 相似文献
20.
André Eggen Mathieu Gautier Alain Billaut élisabeth Petit Hélène Hayes Pascal Laurent Catherine Urban Martha Pfister-Genskow Ken Eilertsen Michael D Bishop 《遗传、选种与进化》2001,33(5):543-548
A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents. 相似文献