484 Constructing the BAC Contig for the Evergreen Gene Region in Peach [Prunus persica (L.) Batsch]

Evergreen genotypes of peach [Prunus persica (L.) Batsch] have been identified in Mexico, where terminal growth on evergreen trees is continuous under favorable environmental conditions. This evergreen trait in peach is controlled by one single gene (evg), and this evergreen condition is homozygous recessive. Four dominant AFLP markers, EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC, were found to be tightly linked to the evg locus at 1 cM, 4.6 cM, 5.8 cM, and 11 cM, respectively. All four markers were sequenced and identified. A peach BAC library was constructed by using the pBeloBAC11 vector for building the physical map for the evg gene. This library represents four times the coverage of the peach genome with the average insert size of 50 to 70 kb. The EAT/MCAC AFLP marker fragment was used for screening the peach BAC library. A single BAC clone, 18F12, was confirmed to contain this fragment. The final BAC contig for this evg gene region and the potential homology between peach and Arabidopsis thaliana will be presented and discussed.

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Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/929472 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Andrea R. Gschwend ◽

Qingyi Yu ◽

Paul Moore ◽

Christopher Saski ◽

Cuixia Chen ◽

...

Keyword(s):

Y Chromosome ◽

Restriction Enzyme ◽

Sex Chromosomes ◽

Physical Map ◽

Bac Library ◽

Specific Region ◽

Average Insert Size ◽

Insert Size ◽

Male And Female ◽

Bac Libraries

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzymeBstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzymeEcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yhchromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes.

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Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research

Genome ◽

10.1139/g99-099 ◽

2000 ◽

Vol 43 (1) ◽

pp. 199-204 ◽

Cited By ~ 32

Author(s):

Junqi Song ◽

Fenggao Dong ◽

Jiming Jiang

Keyword(s):

Bacterial Artificial Chromosome ◽

Physical Map ◽

Molecular Cytogenetics ◽

Bac Library ◽

Artificial Chromosome ◽

Average Insert Size ◽

Chromosome Identification ◽

Insert Size ◽

Potato Genome ◽

Bac Clones

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23 808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes. Key words: potato, BAC library, chromosome identification, physical mapping, molecular cytogenetics.

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Construction of a garlic BAC library and chromosomal assignment of BAC clones using the FISH technique

Genome ◽

10.1139/g03-012 ◽

2003 ◽

Vol 46 (3) ◽

pp. 514-520 ◽

Cited By ~ 8

Author(s):

Hye-Ran Lee ◽

Eun-Mi Eom ◽

Yong-Pyo Lim ◽

Jae-Wook Bang ◽

Dong-Hee Lee

Keyword(s):

Allium Sativum ◽

Bac Library ◽

Chromosomal Assignment ◽

Fish Analysis ◽

Average Insert Size ◽

Insert Size ◽

Bac Clone ◽

Fish Technique ◽

Bac Clones ◽

Cytogenetic Studies

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 pBAC1SACB1 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 (~100 kb) BAC clone on the long arm of garlic chromosome 7. The other BAC clone, GBC2-4d (~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.Key words: garlic, chromosomal assignment, Allium sativum L., FISH technique, BAC library.

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A4Mb High Resolution BAC Contig on Bovine Chromosome1q12and Comparative Analysis With Human Chromosome21q22

Comparative and Functional Genomics ◽

10.1002/cfg.476 ◽

2005 ◽

Vol 6 (4) ◽

pp. 194-203 ◽

Cited By ~ 8

Author(s):

Cord Drögemüller ◽

Anne Wöhlke ◽

Tosso Leeb ◽

Ottmar Distl

Keyword(s):

Positional Cloning ◽

Physical Map ◽

Dominant Gene ◽

Bac Library ◽

Exact Order ◽

Bac Contig ◽

Nucleotide Sequence Data ◽

New Genes ◽

Human Genes ◽

First Time

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genesKRTAP8P1andCLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed thein silicomapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine–human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle. The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned Accession Numbers AJ698510–AJ698674.

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Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/476723 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Jinke Lin ◽

Dave Kudrna ◽

Rod A. Wing

Keyword(s):

Camellia Sinensis ◽

Bac Library ◽

Gc Content ◽

Artificial Chromosome ◽

Tea Plant ◽

Average Insert Size ◽

Plant Genomics ◽

Insert Size ◽

Sequence Class ◽

End Sequences

We describe the construction and characterization of a publicly available BAC library for the tea plant,Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.

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Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

BioMed Research International ◽

10.1155/2013/587493 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Changqing Liu ◽

Yuo Guo ◽

Taofeng Lu ◽

Hongmei Wu ◽

Risu Na ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Blood Cells ◽

Large Scale ◽

Bac Library ◽

Artificial Chromosome ◽

Genetic Dissection ◽

Average Insert Size ◽

Miniature Pig ◽

Insert Size ◽

Statistical Probability

Bacterial artificial chromosome (BAC) libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa), using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

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A Bacterial Artificial Chromosome Contig Spanning the Major Domestication Locus Q in Wheat and Identification of a Candidate Gene

Genetics ◽

10.1093/genetics/164.1.311 ◽

2003 ◽

Vol 164 (1) ◽

pp. 311-321 ◽

Cited By ~ 3

Author(s):

Justin D Faris ◽

John P Fellers ◽

Steven A Brooks ◽

Bikram S Gill

Keyword(s):

Physical Map ◽

Bac Library ◽

Wheat Chromosome ◽

Artificial Chromosome ◽

Physical Distance ◽

Bac Contig ◽

Complete Sequencing ◽

Bacterial Artificial Chromosome Contig ◽

Q Gene ◽

Similarity Searches

Abstract The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of ∼300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q.

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A novel high-accuracy genome assembly method utilizing a high-throughput workflow

10.1101/2020.11.26.400507 ◽

2020 ◽

Author(s):

Qingdong Zeng ◽

Wenjin Cao ◽

Liping Xing ◽

Guowei Qin ◽

Jianhui Wu ◽

...

Keyword(s):

High Throughput ◽

Genome Assembly ◽

Large Scale ◽

Sequence Data ◽

Physical Map ◽

Bac Library ◽

High Accuracy ◽

Biological Research ◽

Bac Clone ◽

Assembly Method

AbstractAcross domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

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Construction of a Populus tremuloides Michx. BAC library

Silvae Genetica ◽

10.1515/sg-2008-0010 ◽

2008 ◽

Vol 57 (1-6) ◽

pp. 65-69 ◽

Cited By ~ 4

Author(s):

M. Fladung ◽

H. Kaufmann ◽

T. Markussen ◽

H. Hoenicka

Keyword(s):

Genome Size ◽

Populus Tremuloides ◽

Random Sampling ◽

Aflp Marker ◽

Bac Library ◽

Male Parent ◽

Haploid Genome ◽

Average Insert Size ◽

Insert Size ◽

Bac Clones

Abstract We have constructed an aspen (Populus tremuloides Michx., line Turesson141) BAC library containing 55,296 clones in total. A random sampling of 86 BAC clones indicated an average insert size of 76 kb with a range of 20 to 160 kb. Twelve percent of the BAC clones in the library have an insert size larger than 100 kb. Based on an estimated genome size for Populus of 500 Mbp, library coverage is about 8 haploid genome equivalents. This library will be screened using AFLP marker identified before co-segregating with gender in a P. tremula x P. tremuloides progeny, where Turesson141 was the male parent.

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Integrative Physical and Genetic Mapping of the Chickpea Genome for Fine Mapping and Analysis of Agronomic Traits

10.32747/2010.7592122.bard ◽

2010 ◽

Author(s):

Hongbin Zhang ◽

Shahal Abbo ◽

Weidong Chen ◽

Amir Sherman ◽

Dani Shtienberg ◽

...

Keyword(s):

Ssr Markers ◽

Dna Markers ◽

Agronomic Traits ◽

Physical Map ◽

Ascochyta Blight ◽

Genetic Maps ◽

Average Insert Size ◽

Marker Assisted Breeding ◽

Insert Size ◽

A Genome

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.

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