Ization of eric primer allowed to discriminate

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ization of ERIC primer allowed to discriminate between the two strains. Similar results were obtained by McManus and Jones (1995a) using other Rubus strains. Our results are in line also with Kim et al. (1995) and Momol et al. (1997), founding two Rubus subgroups by using the BIO- LOG system, and RAPD fingerprinting respectively. How many lineages exist in E. amylovora strains isolated from Rubus spp. would deserve further studies. The RFLP analysis of the Pst I fragment of the pEA29 plasmid pointed out a higher variability in its length than that previously observed by Lecomte et al. (1997). These authors found out three groups of E. amylovora strains according to the length of the lar- ger of the fragments observed after the digestion with Msp I and Sau 3A enzymes. The present study showed that the length of the larger fragment spans from around 160 bp to nearly 400 bp. An example of such a relevant variability is given by two strains from Rubus spp., each one showing a distinct pattern. Similar results were already obtained by Schnabel and Jones (1998) by observing that the expected band of 307 bp varied in length among the different strains tested. Thus, a clear-cut grouping of E. amylovora strains using this technique would not seem reliable. The relevant variability found in the length of the plasmid fragment was explained by a variation in the number of copies of a SSRs of 8 bp, GAATTACA. Two to 15 SSR units have been described in E. amylovora so far (Kim and Geider 1999; Jock et al. 2003; Ruppitsch et al. 2004). Strain ISF-WC31 from the USA was the fourth strain found so far with two SSR units only. All other strains with that specific SSR number were from E. amylovora PD2915 ( Amelanchier sp.) E. amylovora BC199 ( Rubus sp.) E. amylovora BC201 (Rubus sp.) E. amylovora PD103 ( Rubus sp.) E. amylovora BC204 ( Rubus fruticosus ) E. amylovora Ea321 ( Pyrus communis ) E. amylovora NCPPB683 ( Pyrus communis ) Figure 4 Cladogram based on 16S rDNA nucleotide sequence of Erwinia amylovora strains from Amelanchier sp., Rubus spp. and Pyrus communis inferred by means of Clustal W multiple sequence alignment. The branch lengths are not proportionate to sequence divergence. Neighbour joining was chosen as clustering algorithm. m 400 300 200 1 2 3 4 5 6 7 8 m Figure 5 Representative cleavage patterns of the PCR amplification products from plasmid pEA29 of Erwinia amylovora strains after diges- tion with Sau 3A enzyme. M: molecular size marker, 100-bp ladder (Promega); lane 1, E. amylovora PD 2913 (pattern A); lane 2, IVIA 1951-2 (pattern B); lane 3 NCPPB 683 (pattern C); lane 4, PD 4071 (pattern D); lane 5, PD 3368 (pattern E); lane 6, NCPPB 2293 (pattern F); lane 7, NCPPB 2292 (pattern G); lane 8, PD 2915 (pattern H). m 126 222 1 2 3 4 5 6 7 Figure 6 Acrylamide gel showing the detection of single-sequence DNA repeat units in Erwinia amylovora strains after the PCR amplifica- tion using primers RS1 and RS2c; m, molecular base pairs size marker pGEM (Promega); lane 1, E. amylovora H 905; lane 2, PD 3368; lane 3, ICMP 13415; lane 4, ICMP 13417; lane 5, H 902; lane 6, ICMP 13414; lane 7, PD 3891. The band lighter than 222 bp shows the variability in the number of the SSR units.
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  • Spring '08
  • Devartanian
  • DNA, DNA sequencing, Restriction enzyme, Greece Greece Greece Greece Greece Greece Greece Greece Hungary Hungary Hungary Hungary Hungary Hungary Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy USA USA Italy Italy Italy Italy, E. amylovora

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