doi:10.1016/S0168-9452(02)00038-9 
Copyright © 2002 Elsevier Science Ireland Ltd. All rights reserved.
Suppression subtractive hybridization to enrich low-abundance and submergence-enhanced transcripts of adventitious root primordia of Sesbania rostrata
Mercè Caturla1, Cristian Chaparro, Katrien Schroeyers and Marcelle Holsters
, 
Departement Plantengenetica, Vakgroep Moleculaire Genetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Received 10 December 2001;
revised 22 February 2002;
accepted 22 February 2002.
Available online 20 March 2002.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Abstract
Upon waterlogging, the tropical legume Sesbania rostrata activates dormant meristems of stem-located root primordia, leading to adventitious root outgrowth. By suppression subtractive hybridization (SSH), cDNA clones were collected that were enriched for low-abundant and water-induced transcripts. From 192 sequenced SSH clones, ranging from 64 to 1064 bp in size, 66 unigenes were obtained. Complex cDNA probes were generated to screen cDNA macro-arrays. Sequence homologies of putative up-regulated unigenes and of non-induced unigenes will be briefly discussed.
Author Keywords: Dormant root meristem; Root nodule initiation; Suppression subtractive hybridization
Fig. 1. Differential screening. (A) Macro-array hybridized with subtracted forward and reverse cDNA probes. The forward subtracted probe was made using root primordia RNA (control) as driver and root primordia RNA previously immersed in water for 4 h (4 h) as tester, whereas the reverse subtracted probe was made using the 4-h probe as driver and the control as tester. The 192 clones were arrayed in triplicate, in eight vertically placed groups of 6 columns×4 rows each. Brackets indicate the triplicates. (B) Macro-array of the 26 up-regulated unigenes from the first screening hybridized with subtracted forward and reverse cDNA probes and with control and 4-h cDNA probes. For the second hybridization, the 26 cDNA samples were arrayed in two groups of 3 columns×4 rows, plus two additional spots at the top (right). A set of four ubiquitin controls were arrayed on either side (bottom) of the samples and three GFP spikes as well. GFP, green-fluorescent protein; Ub, ubiquitin.
Table 1. Quantitative data from the array hybridization of selected clones with total RNA-derived probes presented in Fig. 1B
Background threshold is calculated as twice the standard deviation of the background signal. Any signal minus background above this level is considered as significant. The background was calculated by averaging the intensities of the pixels on the border and multiplying by the total pixels of each cell. Data for each arrayed clone was obtained by averaging the intensity of the three repeated spots. The standard deviation of the three spots pertaining to a clone was used to detect artefacts or missing spots. The values correspond to the average of the three spots present on the filter for each clone and are corrected with the local background. Values are expressed as arbitrary intensity units.
Table 2. cDNA sequences of 66 unigenes isolated by SSH
All 25 up-regulated clones are shown. Clones with homologies to BLAST-expected values lower than 1.0e−12 are listed as significant homologies; for the non-induced genes, clones with homology to sequences of unknown functions and clones that did not share significant homologies with known sequences are not shown and correspond to: SSH-31 (AJ301763), SSH-42 (AJ301774), SSH-51 (AJ301783), SSH-53 (AJ301785), SSH-54 (AJ301786), SSH-55 (AJ301787), SSH-56 (AJ301788), SSH-57 (AJ301789), SSH-58 (AJ301790), SSH-59 (AJ301791), SSH-60 (AJ301792), SSH-61 (AJ301793), SSH-63 (AJ301795), SSH-64 (AJ301796), SSH-65 (AJ301797) and SSH-66 (AJ301798). CDS, coding sequence.
Abstract
Purchase PDF (188 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (12)
Purchase PDF (483 K)
