The Use of EcoTILLING for the Genetic Improvement of Jatropha curcas L.

Abstract

Distinguishing adaptive variation from neutral mutations are becoming key issues to understand heritable phenotypic changes and to carry out functional genomics. Genetic diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and the population histories that have played a role in the evolution of tree species, such as Jatropha. To efficiently identify SNPs, a number of different techniques have been developed, which all have their limitations. Thus, there is an increasing demand for high-throughput genotyping technologies to increase our ability to determine rare nucleotide differences for traits of economical interest. Reverse genetics approaches rely on the detection of differences in target sequences to identify allelic variations in natural or mutant populations and in the context of functional genomics. EcoTILLING, a variation of Targeting Induced Local Lesions IN Genomes (TILLING), allows high-throughput analyses of natural genetic diversity in plants, particularly in species with limited genetic diversity such as Jatropha curcas. To achieve certain breeding goals in J. curcas, the use of germplasm from different regions is necessary. Understanding the population structure of J. curcas is challenging due to limited genetic variability and information on phylogenetic relationships between accessions of J. curcas and related species. In fact, the genetic map of J. curcas is not well-developed and research on molecular markers is just expanding that could be used to clearly distinguish world wide accessions. Therefore, a resource database of SNPs in J. curcas will give researchers and breeders a tool for answering questions related to population structure, adaptation, and gene function. The development of cultivars of J. curcas by conventional breeding will profit largely from biotechnological support (pathogen-free accessions with specific traits, non-toxic, high yielding varieties). An in vitro germplasm collection of 1,200 accessions from 14 countries was established to (1) conserve valuable genetic resources, (2) survey genetic variation and (3) serve as starting material for genetic improvement with different breeding goals.

Four different pooling strategies were used to increase throughput discovery of homozygous and heterozygous SNP variations. Variations were analyzed both within a single tree (heterozygous) as well as between individual trees and reference samples. Due to the reported low variation between Jatrophaaccessions and the large size of the collection, a 8 × 8 pooling strategy was ultimately chosen to estimate the level of variations among 12 selected genes. EcoTILLING was found to be more efficient for large-scale studies of genetic variation and to investigate the function of candidate genes in J. curcas, compared to RAPD, SSR and AFLP.