Mapping and gene expression associated with the process of organogenic competence acquisition in tomato (Solanum lycopersicum L. cv Micro-Tom)

Author(s)

Mariana da Silva Azevedo

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Centro de Energia Nuclear na Agricultura (CENA/STB)
• Defense date: 2012-09-25

Examining board members

Lazaro Eustaquio Pereira Peres; Maria Helena de Souza Goldman

• Advisor: Lazaro Eustaquio Pereira Peres

Abstract

The dominat allele Rg1 is related to the greater in vitro regeneration capacity of Solanum peruvianum L. The Rg1 gene is required for shoot formation from roots and others explants and was mapped on chromosome 3 between BETA-CAROTENE HYDROXYLASE (CrtR-b) and PHYTOENE SYNTHASE (PSY1) genes. Allelic variants are also known for the genes CrtR-b and PSY1. white flower (wf) is the allele of CrtR-b that produces white petals and yellow flesh (r) is the allele of PSY1 that produces yellow fruits. The alleles wf and Rg1 with r were introgressed into the Micro-Tom cultivar (Solanum lycopersicum L). Through the sequential transfer of explants from SIM to BM and RIM to SIM, we found that MT-Rg1 reduces the time required for the induction of shoots in one day (6 days on SIM for MT), due the reduction of the time required for competence acquisition in one day (2 days to MT). It was obtained 30 putative recombinants from crossing Rg1/rg1 Wf/wf x rg1/rg1 wf/wf. The recombinant lines were scored based on the presence of white petals (wf allele\'s effect) and increased shoot branching (Rg1 allele\'s effect). All putative recombinant lines were tested in vitro for their capability to form shoots in SIM and roots in RIM. Thus, 7 recombinant lines were selected and used to fine mapping the region where RG1 gene is located. CAPS and SCAR markers designed to S. pennellii were tested to fine-mapping, using as template genomic DNA samples from S. pennellii, S. peruvianum, Micro-MsK, MT, MT-Rg1, MT-wf and the 7 recombinant lines. These results confirmed that the molecular markers designed for S. pennellii can be successfully used for S. peruvianum. These analyzes also suggest that the introgressed segment of MT-Rg1 is located between the P5 marker and the CrtR-b gene. Within this region, there are 136 genes, including the GRAS 10 gene whichis the main candidate to RG1 gene function. In addition, we performed RNA-Seq analysis (SOLiD platform) to identify genes differentially expressed in MT and MT-Rg1. It was observed more down-regulated than up-regulated genes in the competence acquisition stage. Due to the large number of differentially expressed genes, some parameters were used for selection of genes that would have their expression validated by qRT-PCR in an in vitro regeneration test. Five genes among 361 genes differentialy expressed were selected to test their expression. Of these 5 genes, GRAS 10 and Serine / threonine protein phosphatase 7 were the most up-regulate genes and showed to be closely related to the stage of competence acquisition. Since there are many GRAS genes, we performed a phylogenetic analysis to identify homologous genes. Based on the phylogenetic analysis, GRAS 10 was identified as homologous to SCL8, a gene already identified in Arabidopsis. However, little is known about SCL8. Thus, it is still necessary to confirm the RG1 gene identity. This approach will provide a better understanding of competence acquisition process. (AU)

• FAPESP's process: 09/13297-6 - Characterization of a gene affecting the competence for organ formation in tomato Solanum lycopersicum L. cv Micro-Tom)
• Grantee: Mariana da Silva Azevedo
• Support Opportunities: Scholarships in Brazil - Master