Construção de um mapa funcional em seringueira (Hevea brasiliensis)

Author(s)

Carla Cristina da Silva

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2014-06-18

Examining board members

Anete Pereira de Souza; Mário Luiz Teixeira de Morais; Maria Bernadete Lovato; Ananda Virginia de Aguiar; Flavio Antonio Maës dos Santos

• Advisor: Anete Pereira de Souza

Abstract

Rubber tree (Hevea brasiliensis), native species of the Amazon, is world¿s major source of natural rubber. Rubber tree breeding programs have been fundamental for the selection of desirable traits. However, the breeding cycle is time consuming (around 30 years), which makes the development of new techniques for early evaluation a necessity. cDNA libraries and Expressed Sequence Tags (ESTs) are very important tools in molecular biology: they enable the identification of genes preferentially expressed in tissues or cellular types and are also a valuable resource of polymorphic markers, powerful instruments for genotyping and molecular mapping. The use of EST-derived markers allows the construction of functional maps, wherein expressed genes or regions near genes are positioned. This type of mapping is important for gene-trait association studies and candidate genes identification. The present study aimed at the construction of cDNA libraries from different tissues (panel, latex and leave) and treatments (cold exposure and Microcyclus ulei controlled infection) of rubber tree for the development of EST sequences and gene-targeted molecular markers, to raise the saturation of a microsatellite-based integrated genetic map previously constructed in our laboratory, in which 18 quantitative trait loci (QTLs) related to growth traits were identified. Sequencing of 10.464 clones generated 8,551 high quality ESTs that were clustered into 5,211 unigenes. Among these, 3,582 (68.7%) showed similarity to a hypothetical or expressed protein. A total of 173 EST-SSR and 43 SNP markers were developed for H. brasiliensis. 150 SSRs (87%) could be associated with functional genes, and 98.8% were transferred to other Hevea species, suggesting that the genus is a complex formed by different species. The SNP markers were identified in 13 ESTs that showed similarity to stress response, development and latex biosynthesis proteins. Six sequences were highly abundant in the cold exposure libraries and expression analyses demonstrated that five sequences were up-regulated during the exposure, with emphasis to two sequences with more than 70-fold increase in expression. From the developed EST-SSRs, 46 were genotyped in the segregating F1 population comprised of 270 plants. These markers were added to the genetic map, which know contains a total of 330 markers. The OneMap software was used for the map construction that now has 3,068.9 cM and 22 linkage groups. Five loci were mapped into QTLs, and transcripts of three of them present similarity to proteins involved in stress response and developmental processes. These loci may be candidate genes for studies related to rubber tree growth traits. To our knowledge, this is the first work in rubber tree that combines analyses of ESTs from different tissues and treatments, and to analyze sequences under cold stress, in several H. brasiliensis genotypes. The new positioned markers may help in the identification of genes of interest and QTLs for other agronomic important traits. The several gene-targeted markers developed here will be used in the mapping and positioning of possible genes in other mapping populations that are now being evaluated at Genetics and Molecular Analysis Laboratory (AU)