Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

Isidore, Edwige; Scherrer, Beatrice; Bellec, Arnaud; Budin, Karine; Faivre-Rampant, Patricia; Waugh, Robbie; Keller, Beat; Caboche, Michel; Feuillet, Catherine; Chalhoub, Boulos

doi:10.1007/s10142-004-0127-9

Cited by 44 publications

(36 citation statements)

References 19 publications

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“…4). Using this procedure, it may become possible to compare multiple genomic regions, among three cultivated barley accessions ('Morex'; Yu et al 2000, 'Cebada capa';Isidore et al 2005, 'Haruna Nijo'; the present study). This may enable the cloning and evaluation of allelic variation of particular gene(s), and the haplotyping analysis of defined chromosomal regions surrounding genes of importance, in multiple genomic backgrounds.…”

Section: Discussionmentioning

confidence: 97%

“…Although the library reported by Yu et al (2000) contained 313,344 gridded clones (6.3 haploid genome-equivalents), the library constructed by Lapitan et al (1997) consisted of only 10,750 clones with an average insert size of 95 kb, and was estimated to be less than one genome-equivalent. Recently, Isidore et al (2005) have constructed a barley cv. 'Cebada capa' BAC library, and adopted a pooling strategy for rapid, cost-reduced screening to obtain targeted clones.…”

Section: Introductionmentioning

confidence: 99%

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Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library from the Japanese Malting Barley variety 'Haruna Nijo'

et al. 2007

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Cultivated barley (Hordeum vulgare L.) is well known as one of the most widely cultivated crops in the world and as an extensively studied plant species in the field of genetics. In recent years, despite its very large genome size (ca. 5,000 Mb), the research resources needed for barley genomic studies have become available, including a large number of expressed sequence tags (ESTs). These have been widely used for barley genome analyses, such as DNA marker-generation and the construction of microarrays. However, the availability of a large-insert genomic library, which is also essential for genomic studies, has been relatively limited in the barley research community. We described here the construction and characterization of a barley bacterial artificial chromosome (BAC) library, using the Japanese malting barley variety 'Haruna Nijo'. The BAC library consisted of 294,912 clones arrayed in 768 384-well microtiter plates. The average size of each cloned insert was estimated to be 115.2 kb, with approximately 0.5% of the clones lacking inserts. Chroloplast DNAs were present in about 1.7% of the library. Thus, the genomic coverage of the 'Haruna Nijo' BAC library was estimated to be about 6.6 genome-equivalents. In order to rapidly identify specific BAC clones, we developed a screening strategy that combined PCR analysis of pooled BAC DNAs with colony hybridization. Using this screening scheme, we investigated the genomic coverage of this BAC library, using 13 locus-specific ESTs and a sequence-tagged site marker. By screening the whole library with individual markers, we identified an average of 5.1 clones per marker. This screening scheme also enabled us to rapidly construct a physical contig spanning a region of approx. 190 kb around the HvBRI1 locus, where the mutation responsible for the semi-dwarf plant type 'uzu' is located. These results indicate that the 'Haruna Nijo' BAC library will be useful for barley genomic studies.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library from the Japanese Malting Barley variety 'Haruna Nijo'

et al. 2007

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“…As the B. napus genome is highly duplicated, to avoid artifacts related to simultaneous PCR amplification of homologous fragments and to directly access the genomic region of the mutant allele Bn-clg1A-1D, we constructed a pooled BAC library of the line 'Primor-Clg' as described by Isidore et al (2005). Screening of the library with PCR markers linked to Bn-clg1A-1D identified three BAC clones that contain this mutant allele.…”

Section: Identification Of the Causal Mutation By Comparison Of The Mmentioning

confidence: 99%

“…To isolate the mutant allele Bn-clg1A-1D, we constructed a pooled large insert library for Primor-Clg cultivar following the procedures described by Isidore et al (2005). Specific PCR markers tightly linked to Bn-clg1A-1D were then used to screen the pools of BAC clones of the library to identify the BAC clone "Clg_H1B_P13a_3_J1" containing the mutant allele.…”

Section: Construction and Screening Of Ordered And Pooled Bac Librariesmentioning

confidence: 99%

A Dominant Point Mutation in a RINGv E3 Ubiquitin Ligase Homoeologous Gene Leads to Cleistogamy inBrassica napus

Arnaud

Belcram

et al. 2012

The Plant Cell

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In the allopolyploid Brassica napus, we obtained a petal-closed flower mutation by ethyl methanesulfonate mutagenesis. Here, we report cloning and characterization of the Bn-CLG1A (CLG for cleistogamy) gene and the Bn-clg1A-1D mutant allele responsible for the cleistogamy phenotype. Bn-CLG1A encodes a RINGv E3 ubiquitin ligase that is highly conserved across eukaryotes. In the Bn-clg1A-1D mutant allele, a C-to-T transition converts a Pro at position 325 to a Leu (P325L), causing a dominant mutation leading to cleistogamy. B. napus and Arabidopsis thaliana plants transformed with a Bn-clg1A-1D allele show cleistogamous flowers, and characterization of these flowers suggests that the Bn-clg1A-1D mutation causes a pronounced negative regulation of cutin biosynthesis or loading and affects elongation or differentiation of petal and sepal cells. This results in an inhibition or a delay of petal development, leading to folded petals. A homoeologous gene (Bn-CLG1C), which shows 99.5% amino acid identity and is also constitutively and equally expressed to the wild-type Bn-CLG1A gene, was also identified. We showed that P325L is not a loss-of-function mutation and did not affect expression of Bn-clg1A-1D or Bn-CLG1C. Our findings suggest that P325L is a gain-of-function semidominant mutation, which led to either hyper-or neofunctionalization of a redundant homoeologous gene.

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“…Positive BAC clones were validated individually by PCR amplification using the primer pairs used for probes synthesis. Two additional BAC libraries named Ttu-B-TTD140-ng (HindIII restriction) and Ttu-B-TD140e-ng (EcoRI restriction) were constructed from the same plant material using a nongridded BAC library protocol based on Isidore et al (2005) with the following modifications: growth of pooled BAC clones in liquid LB medium, BAC pool DNA amplification by the Whole Genome Genomiphi.v2 phi29 enzyme kit (GE Healthcare) instead of DNA extraction, and use of secondary pooling steps to identify positive clone coordinates with less screening effort after clone picking. HMW DNA extractions and sizing steps were processed as for the first classical BAC library.…”

Section: Construction Of Three Bac Libraries From Ttd140mentioning

confidence: 99%

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness

et al. 2016

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The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular b-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of b-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating b-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a b-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in b-diketone biosynthesis, demonstrating a gene cluster also in the b-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

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Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

Cited by 44 publications

References 19 publications

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library from the Japanese Malting Barley variety 'Haruna Nijo'

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library from the Japanese Malting Barley variety 'Haruna Nijo'

A Dominant Point Mutation in a RINGv E3 Ubiquitin Ligase Homoeologous Gene Leads to Cleistogamy inBrassica napus

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness

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