Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

Authors: Koike, Michiya; Kawaura, Kanako; Ogihara, Yasunari; Torada, Atsushi

Source: Genome, Volume 49, Number 5, May 2006 , pp. 432-444(13)

Publisher: NRC Research Press

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Abstract:

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.Key words: common wheat, simple sequence repeat (SSR), PCR screening, LTR-retrotransposon, TAC clone.

Les auteurs ont développé une méthode PCR en 2 étapes, analogue à une PCR suppressive, pour isoler aisément des microsatellites (SSR) chez le blé (Triticum aestivum L.). Cette approche ne nécessite ni le criblage de banques génomiques ni de processus d'enrichissement sélectif. Les auteurs ont produit 131 paires d'amorces basées sur des séquences de microsatellites isolés non seulement d'ADN génomique mais également de clones TAC. Il a été démontré que 34 de ces 131 microsatellites étaient polymorphes au sein de huit génotypes de blé. Quatre des 34 marqueurs polymorphes étaient issus de clones TAC, ce qui indique que cette approche rend possible le développement ciblé de marqueurs uniques au sein de grandes banques génomiques constituées de clones BAC ou PAC. Plusieurs clones contenant des microsatellites présentaient une similitude avec un segment LTR de plusieurs rétrotransposons identifiés chez divers génomes de hordées. La plupart de ces microsatellites produisaient des profils d'amplification en dégradé, ce qui suggère que plusieurs microsatellites détériorés. provenant d'ADN répétitif et particulièrement les LTR-RT, existent dans le génome du blé tendre.Mots clés : blé tendre, microsatellite (SSR), crible PCR, rétrotransposon à LTR, clone TAC.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: May 1, 2006

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  • From its inception in 1957, this international cytogenetics journal has catered to the research areas of the members of the Genetics Society of Canada; traditionally, these have included agriculture, entomology, genetics/cytogenetics, and evolutionary mechanisms. The contents of the journal have evolved as contributors developed new technologies and interests. A 20-member Editorial Board is composed of scientists from around the world. Reviews and commentary from respected experts are often featured.
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