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Abstract

The goal of plant breeding is to combine naturally occurring genetic variation between
species, strains and varieties to create superior varieties. Being able to locate the genomic
region responsible for the trait of interest accelerates this process, because it allows for
more direct selection of the breeding material.
Traditionally, standard genetic mapping a trait of interest in crops required established
sets of molecular markers able to distinguish the parental lines to genotype the progeny.
Marker development and genotyping individual plants is tedious and costly.
We developed a method for genetic mapping by 2nd-generation sequencing, which does
not require prior knowledge of molecular markers and will also directly yield the causal
polymorphism. It is based on bulked segregant analysis of pools of plants. By sequencing
two samples, one lane each on the Illumina Genome Analyzer II, we mapped the causal
nucleotide change for a recessive virus resistance in a segregating population of two non-
reference indica rice strains within a few weeks. While a reference genome is necessary,
sequence information of the parental genomes is not.
The current resolution of genetic mapping projects often only allows one to define a
region of the genome responsible for the trait rather than attributing it to a single gene.
However, researchers are often able to identify one or several candidate genes in those
regions. Specifically silencing these genes in vivo and monitor the effect is an effective
way of testing the candidates. This can be achieved by post-transcriptional gene silencing
(PTGS). Silencing genes with Artificial miRNAs (amiRNAs) – designed to target one or
several genes of interest – have been shown to be a highly specific approach in plants.
We devised an amiRNA-based strategy for both japonica and indica type strains of
cultivated rice, Oryza sativa. The adoption of this technology to rice will be described.
Besides candidate gene validation, the technology may be used for comparative functional
genomics between different varieties, and for improvement of agronomic performance and
nutritional value.