Volume 5 Supplement 7
Construction and analysis of a leaf cDNA library from cold stressed rubber tree clones
© Da Silva et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Rubber tree [Hevea Braziliensis (Willd. ex Adr. de Juss.) Muell-Arg.], a native species of the Amazon Rainforest in South America, belonging to the Euphorbiaceae family, is world’s major source of natural latex. Brazil, despite being the center of origin and diversity of the species and latex main producer at the end of XIX century, nowadays imports around 70% of the rubber consumed in the country. Although the Amazonian basin provides optimal conditions for rubber plantation, the occurrence of South American leaf blight (SALB) disease, caused by the ascomycete Microcyclus ulei, limits the rubber production in this region. Therefore, rubber plantation has been extended to suboptimal areas that are prominently located in northeast India, Vietnam, southern China and southern plateau of Brazil. Besides the new conditions for crop development, these new areas present stress conditions like low temperatures, dry periods and wind. It was shown that low temperatures affect the development and latex production of rubber trees. Breeding programs have been searching for clones adapted to these suboptimal areas. A complete cycle of breeding and selection takes at least 20 years to succeed in obtaining a superior clone. As such, development of new methods for early evaluation is essential to reduce and optimize the breeding management. Aiming to help in the development of these methods, in this work, we have constructed a leaf cDNA library to study ESTs (Expressed Sequence Tags) from cold stressed rubber trees. The originated sequences from this library can be used to create an ESTs databank and will provide genetic knowledge about how rubber trees cope with chilling stress, being also a valuable source of polymorphic molecular markers, such as SSRs and SNPs. Some of these markers may be associated to chilling tolerance, and early evaluation would be possible in young rubber tree clones.
The young Hevea Braziliensis Muell. Arg. clones GT1, IAN873, PR255 and PB217 were obtained from Agronomic Institute of Campinas (IAC). GT1, PR255 and PB217 clones are genitors of mapping populations of ongoing projects at Molecular Genetics and Analysis Laboratory and IAN873 clone showed chilling tolerance in field. The 24h cold treatment was peformed in a diurnal growth chamber (8°C, 12h photoperiod). The collected leaves were previously wrapped up in tinfoil to prevent transcript redundancy. Leaves were sampled in intervals of 6h, 10h and 24h and total RNA was extracted by LiCl precipitation. Total RNA extracted from clones were pooled in equal amounts, according to time of sampling. The In-fusion SMARTer cDNA Library Construction Kit (Clontech) was used to construct the cDNA library. A sequenced plaque (96 clones) of each sub-library (6h, 10h and 24h) was chosen for a preliminary analysis of the library. Sequences were analyzed and clustered by the DNAStar package (DNAStar, Inc.) and Blast2Go software (http://www.blast2go.org) was used to annotate the contigs and singlets. The resulting ESTs were screened for microsatellite regions using Simple Sequence Repeat Identification Tool (SSRIT) (http://www.gramene.org/db/markers/ssrtool).
Results and discussion
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.