Linkage analysis of EST cDNA clones by using RFLP in the silkworm, Bombyx mori.

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  • カイコにおけるＥＳＴ化したｃＤＮＡクローンのＲＦＬＰによる連関検索
  • カイコ ニ オケル ESTカ シタ cDNA クローン ノ RFLP ニ ヨル レンカン ケンサク

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Abstract

We started the linkage analysis of cDNA clones by using their restriction fragment length polymorphism (RFLP) and examined which strains should be crossed with, how to prepare the individual DNAs, probes, and so on. From the randomly picked-up plaques of 3 day-old embryonic cDNA library, EST-cDNA-clones of which insert size were longer than 500 by were subcloned into plasmid. The DIG ribo-probes were prepared by in vitro transcription and used for the Southern blot analysis. Some clones characterized already were used for screening the strains and the results indicated that p50 and J02 were isogenic. So we decided to use these strains and their cDNA-clones were analyzed. 57% of the clones were found to be useful for RFLP analyses. Sequence data for identification of clones gave new information, firstly, there were many unknowns and five ribosomal-protein-genes and four heat-shock-protein-genes, secondly, most of the clones that gave smear bands had repetitious sequences like Bm1 and Bm2. Linkage analyses of these clones were done by using the segregants of (p50×J02) female and J02 male. Every clone showing heterozygous or homozygous bands could be easily identified that they are linked or not. So this method was named scanning linkage analysis (SLA). Till now 29 cDNA clones were divided into 9 linkage groups and 8 independent clones out of 28 chromosomes.

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