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BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut.

Guimarães PM, Garsmeur O, Proite K, Leal-Bertioli SC, Seijo G, Chaine C, Bertioli DJ, D'Hont A - BMC Plant Biol. (2008)

Bottom Line: The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb.Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Unit, Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil. messenbe@cenargen.embrapa.br

ABSTRACT

Background: Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.

Results: We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.

Conclusion: These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

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GISH of Arachis hypogaea metaphase chromosomes. Somatic metaphases of Arachis hypogaea (subsp. hypogaea var. hypogaea, race Guaycurú) after a) 4'6-diamidino-2-phenylindole (DAPI) counterstaining (blue, shown in black and white), b) Genomic in situ hybridization using genomic DNA from A.duranensis (in green) and A. ipaënsis (red).
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Figure 1: GISH of Arachis hypogaea metaphase chromosomes. Somatic metaphases of Arachis hypogaea (subsp. hypogaea var. hypogaea, race Guaycurú) after a) 4'6-diamidino-2-phenylindole (DAPI) counterstaining (blue, shown in black and white), b) Genomic in situ hybridization using genomic DNA from A.duranensis (in green) and A. ipaënsis (red).

Mentions: Total genomic DNA of A. duranensis (AA genome) and A. ipaënsis (BB genome) when used as probes on the chromosomes of A. hypogaea displayed intense and uniform hybridization patterns onto AA and BB chromosomes of A. hypogaea respectively (Fig. 1). This clear genome discrimination of the chromosome subsets from the corresponding parental genomes in the tetraploids was possible without the need of any unlabelled blocking DNA, which is normally used to avoid cross-hybridization between a specific probe from one genome and homologous DNA sequences from another genome. Counterstained A and B chromosomes show very similar total sizes for the two genomic components (Fig. 1).


BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut.

Guimarães PM, Garsmeur O, Proite K, Leal-Bertioli SC, Seijo G, Chaine C, Bertioli DJ, D'Hont A - BMC Plant Biol. (2008)

GISH of Arachis hypogaea metaphase chromosomes. Somatic metaphases of Arachis hypogaea (subsp. hypogaea var. hypogaea, race Guaycurú) after a) 4'6-diamidino-2-phenylindole (DAPI) counterstaining (blue, shown in black and white), b) Genomic in situ hybridization using genomic DNA from A.duranensis (in green) and A. ipaënsis (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254395&req=5

Figure 1: GISH of Arachis hypogaea metaphase chromosomes. Somatic metaphases of Arachis hypogaea (subsp. hypogaea var. hypogaea, race Guaycurú) after a) 4'6-diamidino-2-phenylindole (DAPI) counterstaining (blue, shown in black and white), b) Genomic in situ hybridization using genomic DNA from A.duranensis (in green) and A. ipaënsis (red).
Mentions: Total genomic DNA of A. duranensis (AA genome) and A. ipaënsis (BB genome) when used as probes on the chromosomes of A. hypogaea displayed intense and uniform hybridization patterns onto AA and BB chromosomes of A. hypogaea respectively (Fig. 1). This clear genome discrimination of the chromosome subsets from the corresponding parental genomes in the tetraploids was possible without the need of any unlabelled blocking DNA, which is normally used to avoid cross-hybridization between a specific probe from one genome and homologous DNA sequences from another genome. Counterstained A and B chromosomes show very similar total sizes for the two genomic components (Fig. 1).

Bottom Line: The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb.Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Unit, Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil. messenbe@cenargen.embrapa.br

ABSTRACT

Background: Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.

Results: We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.

Conclusion: These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

Show MeSH