Using Real-Time PCR to quantify retrotransposon-based insertion polymorphism in the modern polyploid sugarcane cultivars

• Grant number: 13/09391-2
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: September 12, 2013
• End date: December 11, 2013
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Principal Investigator: Marie-Anne Van Sluys
• Grantee: Cushla J Metcalfe
• Supervisor: Karen Aitken
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: CSIRO Plant Industry, Australia

Abstract

Sugarcane is an important crop both in Brazil and in Australia. Modern cultivars have large and highly polyploid genomes, it is estimated that they have 8-10 alleles, depending on the cultivar. For most cultivars, however, the exact genome size is unknown. Sugarcane therefore presents particular difficulties in developing markers for breeding programs. The Retrotransposon-Based Insertion Polymorphism method (RBIP) is a PCR based method used to determine the presence of individual Long Terminal Repeat (LTR)- retrotransposons (a type of transposable element).It has been used successfully in other grass genomes as a DNA marker system but because it is PCR based can only be used in diploid organisms.We have developed a system based on the RBIP method combined with quantitative PCR to estimate the proportion of alleles with the LTR-retrotransposon present at a given loci to create a LTR-retrotransposon profile. Preliminary results suggest that the results are consistent over 14-16 replicates, that a ten-fold difference in template does not give significantly different results, and that patterns of ratio of the number of inserts at 3 loci is variable enough to distinguish different cultivars. We now wish to further this research in two ways 1. to compare the profiles of Brazilian and Australian cultivars and 2. to transfer this method to digital PCR, which is reported to be both more sensitive and precise, but will also allow us to move to a high through-put system. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

METCALFE, CUSHLA J.; OLIVEIRA, SARAH G.; GAIARSA, JONAS W.; AITKEN, KAREN S.; CARNEIRO, MONALISA S.; ZATTI, FERNANDA; VAN SLUYS, MARIE-ANNE. Using quantitative PCR with retrotransposon-based insertion polymorphisms as markers in sugarcane. Journal of Experimental Botany, v. 66, n. 14, SI, p. 4239-4250, JUL 2015. (08/52074-0, 13/09391-2)

METCALFE, CUSHLA J.; OLIVEIRA, SARAH G.; GAIARSA, JONAS W.; AITKEN, KAREN S.; CARNEIRO, MONALISA S.; ZATTI, FERNANDA; VAN SLUYS, MARIE-ANNE. Using quantitative PCR with retrotransposon-based insertion polymorphisms as markers in sugarcane. Journal of Experimental Botany, v. 66, n. 14, p. 12-pg., 2015-07-01. (08/52074-0, 13/09391-2)